Coagulase-negative Staphylococci in Danish blood cultures: species distribution and antibiotic susceptibility

Journal

of Hospital

Infection

(1996)

32,

217-227

Coagulase-negative Staphylococci cultures: species distribution susceptibility

in Danish blood and antibiotic

J. 0. Jarlsv*+, T. Hsjbjergz, C. Busch-Serensen’, J. Scheibel§, J. K. Melled, H. J. J. Kolmos** and D. A. Wandal1f-f *Division of Microbiology, Statens Seruminstitut; f-Department of Clinical Microbiology, National University Hospital (Rigshopitalet), Copenhagen; $Department of Clinical Microbiology, Aalborg Hospital; $Department of Clinical Microbiology, Herlev Hospital, Copenhagen; TDepartment of Clinical Microbiology, Aarhus University Hospital; **Department of Clinical Microbiology, Hvidovre Hospital, Copenhagen; and f+Department of Clinical Microbiology, Bispebjerg Hospital, Copenhagen, Denmark Received

21 September

1995; revised manuscript

accepted 23 November

1995

Summary:

The distribution and antibiotic susceptibility of coagulase-negative staphylococci (CONS) isolated from blood cultures was examined in samples from hospitals covering most of Denmark. A total of 499 CONS isolates were detected in 477 blood cultures from 340 patients and speciated as Staphylococcus epidermidis, 285; Staphylococcus hominis, 61; Staphylococcus haemolyticus, 43; Staphylococcus warneri, 12; Staphylococcus cohnii, 7; Staphylococcus saprophyticus, 4; Staphylococcus capitis, 2 and Staphylococcus Zugdunensis, 1. Seventy-eight isolates could not be identified to species level and six were Micrococcus spp. In 108 (22.6%) blood culture sets, more than one CONS strain were found, as detected by species identification, antibiogram and biotyping. Significantly more blood cultures from patients in university hospitals were drawn from central venous catheters. Comparing university and non-university hospitals, the overall antibiotic susceptibility among CONS was only slightly different, except for methicillin and amikacin. The prevalence of methicillin-resistant strains was 35.1% in the university hospital strains vs. 25.3% in the non-university hospital strains. The overall prevalence of methicillin resistance was 32%. Great geographic variation in both species distribution and antibiotic resistance was observed. The high prevalence of S. epidermidis makes subtyping of this species important. Keywords: antibiograms.

Coagulase-negative

staphylococci;

blood

cultures;

identification;

Introduction

Isolation of coagulase-negative staphylococci (CONS) from blood cultures always raises the question of whether the bacteria represents a true inCorrespondence Juliane Maria

to: Jens Otto Jarlov, Department Vej 22, DK 2100 Copenhagen

0,

of Clinical Denmark.

Microbiology,

Afsnit

9301,

Rigshospitalet,

218

J. 0. Jarlrav et aZ.

fection.lm7 CONS are normal inhabitants of the skin and mucous membranes, but may cause infection in the immunocompromised host, neonates,4.7 intensive-care patients and others especially those with indwelling i.v. devices.lm9 Antibiotic resistance among CONS in Denmark, especially methicillin resistance, seems to follow the same pattern as in other countries”‘” in contrast to methicillin-resistant Staphylococcus auwus, which has become less common, with more than half of cases being imported.” In a previous study dealing with staphylococci in blood cultures from non-university hospitals in two smaller Danish counties,13 the prevalence of methicillin resistance was 40% for Staphylococcus epidermidis and 21% for other CONS species. Furthermore, we found a high number of specimens that grew more than one CONS strain.13 In order to investigate the prevalence of methicillin resistance, resistance to other antibiotics and the extent of growth of multiple CONS strains in Danish blood cultures, we conducted the present study, which included hospitals from five of the 14 Danish counties and Copenhagen Municipality. Materials

and

methods

The following five Danish counties: Northern Jutland, Aarhus, Funen, Roskilde, Copenhagen, representing different parts of Denmark, were included in this study together with Copenhagen Municipality. The investigated regions represent 2.9 million inhabitants out of the total Danish population of 5.2 million. All blood cultures that were CONS positive in a two-month period in the autumn 1992 or winter 1992-i 993 were included. From Roskilde county the collection time was expanded to three months. The blood culture systems used are listed, together with the number of CONS-positive specimens in Table I. It was noted whether the patients had one or more central venous catheters (CVC) and if the blood culture was drawn from a CVC. Species identi$cation and susceptibility testing All positive blood cultures were subcultured locally and either the agar plate or a heavy inoculum from the agar plate was referred to the Staphylococcus Laboratory, Statens Seruminstitut, Copenhagen. In order to detect the possible presence of more than one CONS species or type, a heavy inoculum (to obtain confluent growth) was spread on a blood agar plate and susceptibility testing performed. In addition, a stepwise cultivation was done, and if either different colony morphologies or susceptibility-patterns were observed, each different colony was further tested: (1) species identification was performed with a modified Kloos and Schleifer scheme14 supplemented by an ornithine-decarboxylase test, (2) biotyping was performed by means of Minibact-S,‘” (3) a secondary susceptibility test with semiconfluent growth was performed with penicillin (breakpoint 0.25 pg), methicillin

CONS Table

Roskilde Copenhagen Couyty Copenhagen Mumcqxlity: Bispebjerg Hvidovre Funen County Aarhus County Northern Jutland County Total

in

I. Distribution

Danish

blood

of patients

cultures

219

and blood

cultures

No. of patients

No. of patients with CVC Yes/No/ Unknolvn

No. of blood cultures

36 79

512813 19/40/20

40 158

5:

21 30 67 48 113 477

I 8 11 1

19 2.5 60 44 77 340*

4/l s/o 11/10/4 25/l 3122 2117125 9/52/l 6 75/175/90

* 175 females and 165 males. ‘rhe following blood culture systems u-ere used: ‘Colorbact’: Northern Jutland County. ‘Bactec’: Plarhus County. Roche: system (Statens Seruminstitut): TTunen County. CVC, central venous catheter.

No. of cvcdrawn blood cultures

Roskilde, Bispebjerg.

No. of blood cultures with >l strain

ki

Copenhagen Conventional

No. of strains

7 25

45 128

10 6 21 13 26 108

31 32 88 59 116 116

County, blood

Hvidovre, culture

(8 pg), cephalothin (4 pg), cefuroxime (4 pg), cefotaxime (2 pg), streptomycin (8 pg), gentamicin (4 pg), netilmicin (4 pg), amikacin (4 pg), tetracycline (2 pg), chloramphenicol(6 pg), ciprofloxacin (1 pg), erythromycin (1 pg), fusidic acid (2 pg), rifampicin (0.5 pg), teicoplanin (2 pg) and vancomycin (2 pg) using ‘Neosensitabs’ (Rosco, Taastrup, Denmark).‘” The plates were incubated at 3 5°C and read after 18 h. Methicillin susceptibility was tested on an agar plate with 7.5% NaCl. The latter plates were read after 18 and 48 h and zone-diameter less than 21 mm, or the presence of colonies within the inhibition zone, was used as indicative of methicillin resistance. Identical strains found in different blood cultures from the same patient was included only once in the analysis. Statistical analysis Chi-square test with Yates correction or Fisher’s exact test were used to evaluate differences. PqO.05 was considered significant. Results

A total of 477 blood cultures from 340 patients grew CONS. More than one blood culture (range 2-1.5) were received from 71 patients. Four hundred and ninety-nine different CONS strains were identified and speciated as S. epidermidis, 285; Staphylococcus hominis, 61; Staphylococcus haemolyticus, 43; Staphylococcus warneri, 12; Staphylococcus cohnii, 7; Staphylococcus saprophyticus, 4; Staphylococcus capitis, 2; and one Staphylococcus lugdunensis. Seventy-eight isolates could not be identified to species level and six strains were identified as Micrococcus spp.

220

J. 0. Jarlcav et GE. Table

Percentage resistant strains according to subspecies

II.

Percentage Species

Staphylococcus epidermidis N=285 Staphylococcus hominis N=61 Staphylococcus haemolyticus N=43 Staphylococcus warneri N=l2 Staphylococcus cohnii N=7 Staphylococcus saprophyticus N=4 Staphylococcus capitis N=2 Staphylococcus lugdunensis N=l Micrococcus spp.

resistance

pen

met

str

gen

net

ami

77

41

5

33

5

28

54

18

12

10

0

61

33

23

16

42

0

8

57

71

71

100

0

00 S

S

chl

cry

fus

rif

tip

17

10

28

30

4

5

7

26

8

28

10

0

7

0

20

26

5

33

5

0

9

8

8

0

17

8

0

0

0

0

29

14

57

57

57

57

0

57

43

tet

0

0

0

25

0

0

0

0

0

0

0

0

0

0

0

100

0

0

0

0

R

S

s

s

s

s

s

S

s

s

N=6 Unidentified N=78

67

50

50

17

0

33

17

17

17

17

17

51

15

6

13

1

10

18

3

9

17

0

0

Total

67.5

32.1

8.8

24.2

3.2

21-8

19.4

10.2

24.6

22.4

3-4

5.6

All strains were sensitive to vancornycin and teicoplanin. Pen, penicillin G; met, methicillin; str, streptomycin; gen, gentamicin; net, netilmicin; tetracyclin; chl, chloramphenicol; ery, erythromycin; fus, fusidic acid; rif, rifampicin;

17

ami, amikacin; tet, tip, ciprofloxacin.

More than one CONS strain were found in 108 (22.6%) (two strains in 102 and three in six) blood culture sets from 97 patients. In 78 of these blood cultures, the strains belonged to different CONS-species. In 25 blood cultures, the strains differed in both susceptibility patterns and biotype, but belonged to the same species, mainly S. epidemzidis. Major differences in susceptibility patterns of the same species were found in four blood cultures, and major differences in biotype were found in one blood culture. The antibiotic susceptibility-patterns of the CONS-species are noted in Table II, and the number of strains resistant to one or more antibiotics are listed in Table III. The most resistant species seemed to be S. cohnii followed by S. epidermidis and S. haemolyticus. Significant geographic variation in the species distribution and in antibiotic susceptibility was observed (Tables IV and V). Methicillin resistance ranged from 40% (51/

CONS in Danish Table

III.

The

distribution

Suscew tible. to all

Species Staphylococcus epidermidis N=285 Staphylococcus hominis N=61 Staphylococcus haemolyticus N=43 Staphylococcus %*arneri N=12 Staphylococcus cohnii N=7 Staphylococcus saprophyticus N=4 Staphylococcus capitis N=2 Staphylococcus lugdunensis N=l Micrococcus spp. N=6 Unidentified N=78 * Shown

in Table

of CONS-strains

Number

blood

resistant antibiotics*

of strains

221

cultures to various

resistant

numbers

to various

of the investigated

numbers

of antibiotics

1

2

3

4

5

6

7

8

9

10

11

46

69

37

25

35

32

21

14

6

0

0

0

17

16

15

4

3

4

0

2

0

0

0

9

17

2

2

6

0

2

3

1

0

0

5

4

2

1

0

0

0

0

0

0

0

0

1

0

2

0

0

0

1

1

1

1

0

3

1

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

2

0

0

1

1

1

0

0

0

0

0

25

30

6

6

4

2

3

2

0

0

0

II

128) in Copenhagen County to 21% (24/l 16) in Northern Jutland County (Table V). The p revalence of S. epidermidis ranged from 73% (46/63) in Copenhagen Municipality to 46% (53/l 16) in Northern Jutland County. Those CONS strains, which could not be identified, were more prevalent in Aarhus and Northern Jutland counties than in the Copenhagen area (Table IV). The most pronounced differences in antibiotic susceptibility between the different areas are listed in Table V. In general, the highest frequency of antibiotic resistance was found in the Copenhagen area and in Aarhus county (Table V). No pronounced differences were found in antibiotic susceptibility between university and non-university hospitals, except for methicillin where 35.1% (121/345) were resistant in the university hospitals (Table hospitals and 25.3% (39/l 54) resistant in the non-university V) and amikacin 26% (89/345) vs. 13% (20/154). Eighty-eight blood cultures from 75 patients were drawn through a CVC (Table I). No information about the presence of a CVC could be obtained

J. 0. Jarlev

222 Table

Species

Roskilde County N=45

Staphylococcus epidermis N=285 58 Staphylococcus hominis N=61 13 Staphylococcus haemolyticus N=43 2 Staphylococcus warneri N=12 4 Staphylococcus cohnii N=7 2 Staphylococcus saprophyticus N=4 0 Staphylococcus capitis N=2 0 Staphylococcus lugdunensis N=l 0 Micrococcus spp. N=6 0 Unidentified N=78 20 * Significantly

different

IV.

Percentage

Copenhagen County N=128

distribution Copenhagen Municipality N=63

et al.

of CONS-specied

Funen County N=88

Aarhus County N=59

in Danish

regions

Northern Jutland County N=116

University hospitals N=345

Nonuniversity hospitals N=154

57

73*

66

49

46*

57.7

55.8

15

10

6

9

17

11.0

14.9

12

6

8

14

7

9.8

5.8

2

2

5

2

2

2.3

2.6

2

2

0

2

1

1.7

0.6

0

0

5

0

0

1.1

0

1

0

0

0

1

0.6

0

1

0

0

0

0

0.3

0

1

0

1

2

3

1.4

0.6

8-t

10-t

24~

24$

14.8

17.5

lot (BO.05);

+ vs.$

Significantly

different

(P
concerning the blood cultures from 90 patients. These blood cultures, which were similar to the blood cultures from patients without CVC with regard to number of CONS strains in the blood culture and susceptibilitypatterns, are not included in the calculations concerning CVC. Significantly more blood cultures from patients in university hospitals were CVC-drawn [82/252 (32.5%) vs. 6/90 (6*7%)] and more CONS isolates from patients with CVC were methicillin resistant [54/120 (45%) vs. 67/ 255 (26*3%)] (Table VI). Furthermore, significantly more strains from patients with CVC were S. epidermidis [84/120 (70%) vs. 135/255 (53%), P
CONS in Danish Table

\r.

Percentage

blood

resistant

cultures

strains

according

Percentage Region Roskilde Count) ,v=45 Copenhagen County *V=128 Copenhagen Municipality X=67 Funen County N=X8 Aarhus County N=59 Northern Jutland Count) X=116 University hospitals TV= 345 Non-universit) hospitals N=l54 Total

pen

met

str

f-w

net

223 to regions

resistance

ami

tet

chl

cry

fus

rif

tip

67

29

21

27

2

13

20

4

27

27

2

4

69

40*

14-t

23

2

27t

17

9

32*

27t

5

4

81

39

6

27

8

25

16

1X

31

24

12*

IO

66

31

6

17

1

26

9

7

IS*

19

1

3

63

34

IO

32t

7

25

12

12

20

2.i

2

IO

63

21*

9

16-t

2

13t

20

11

21

161

1*

4

68.7

35.11

9-5

25.6

3.7

25.9*

19.X

II.5

24.4

24.1

4.3

5..i

65.6

25.3-t

7.1

214

I.9

13*

18.2

7.1

24.7

18.2

I.9

5.8

67.7

32.1

8.8

24.3

3.2

21.9

19.3

IO.2

24.5

22.3

3.6

5.6

* Significantly different (PO.01) from -t Significantly different (P
other other and

result in the result in the teicoplanin.

same same

column column

with with

the the

same same

mark. mark.

patients without CVC, but significantly fewer CI/C drawn blood cultures grew more than one CONS-strain; comparing only the blood cultures from the patients known to have a CVC, this difference was the same [lo/88 (11.4%) vs. lo/28 (35.70/o), Table VI] and no significant difference in methicillin resistance among the CONS strains could be found [28/63 (44.4%) vs. 16/33 (48..5%)].

Discussion

Many papers dealing with antibiotic resistance, especially methicillin resistance, in CONS have focused on tertiary care/university hospitals.‘-” A high degree of antibiotic resistance has been found. The skin flora of nontreated individuals has been found to include a vast number of multiply resistant CONS.” Methicillin-resistant CONS were found in a low number on the skin. In a previous paper,13 concerning two small Danish counties, it was noted

present

VI.

The

of central

2 25 228 108

141

number of blood cultures (N=477)

Total

influence

catheters

on CONS

18* 6* 2 2 31

1:: 77

(BO.05).

more

Blood cultures from nonuniversity hospitals (N=133)

(CVC)

123* 82* 26

cultures from university hospitals (N= 344)

Blood

venous

The blood cultures with no information about CVC are not included in the statistical calculations. *Significantly different (RO~OOOOl) from other result in the same row with the same mark. T Significantly different (P
Blood

cultures CVC-drawn Blood cultures not CVC-drawn Unknown CVC not present No information about CVC

CVC

Table

than

cultures

one

isolate.

This

54/120 (45)t 28/63 (44.4)§() 16/33 (48.5)ij lo/24 (41.7) 67/255 (26.3)t 39/124 (31.5)

Methicillin resistant/total isolates (%) 160/499 (32.1)

in blood

difference

is significant

25/141 (17,7)9 10/88 (11.4)$1 10/28 (35.7)s S/25 (20) 58/228 (25.4)s 25/108 (23.1)

Number of blood cultures with >I CONS isolate/total Number of cultures (%) 1081477 (22.6)

t? g c it p -

9

4

w P

CONS in Danish

blood

cultures

225

that growth of multiple CONS strains in blood cultures was a frequent event. The present study was conducted to investigate this phenomenon and to survey antibiotic susceptibility among CONS isolates from different regions in Denmark. Remarkable geographic variation in the prevalence of different CoNSspecies and antibiotic susceptibility was observed. This could not only be explained by differences in hospital categories because all the regions studied, except for Roskilde County, included both university hospitals and smaller hospitals. In a previous Danish study some geographic variability in the prevalence of CONS species and phage-typability was found.18 These findings connected with our results might indicate the occurrence in hospitals of not only a local hospital flora, but also a local community flora from which the local hospital flora is derived. The antibiotic policy in the local area is probably important in selecting resistant CONS-strains in the hospitals.“~” Resistance to methicillin and amikacin were more prevalent among CONS strains from university hospitals compared with the smaller hospitals. Because amikacin is rarely used in Denmark, the rather high incidence of amikacin resistance was surprising. A marked co-variation between amikacin, gentamicin and methicillin was found (data not shown), pointing towards co-selection and this could in part explain the difference between the types of hospitals. However, it should be noted that a large number of the amikacin resistant strains had zone diameters near the breakpoint zone and these strains may not be truly resistant, in vitro. The influence of the presence of a CVC is difficult to evaluate. Not surprisingly, more patients from university hospitals had a CVC. Blood cultures from patients with CVC usually had growth on one single CONS strain, and a high number were methicillin resistant S. epidermidis. This might indicate that these strains were selected in the hospital, had colonized the CVC and, thereby, caused bacteraemia. Two or more CONS strains were frequently found in one blood culture. The significance of a blood culture with growth of multiple CONS strains is a matter for debate. In some instances, it seems reasonable to regard the patient to be truly infected with one or more of these multiple isolates, but the majority of cases should be considered insignificant.13’20 It is important to detect the CONS strain that has the highest degree of antibiotic resistance in order to treat the patient with the correct antibiotics. Several colonies of CONS from the blood culture should, therefore, be included when primary antibiotic susceptibility testing is performed. If only one or two colonies are included the more resistant isolates may be overlooked. Several papers have discussed the method that should be used for the detection of methicillin resistance.21-24 Isolates, which were found to be resistant in our agar diffusion method with 7.5% NaCl, had minimum

J. 0. Jarlev

226

et al.

inhibitory concentrations (MI&) corresponding to 38 mg/L, in a platedilution method using Columbia agar with 4.5% NaCl and an inoculum of approximately 1O6cfu (unpublished data). However, our plates were difficult to read. A NaCl concentration of 795% is probably not optimal with CONS, which grow with some variation on this medium. MIC breakpoints recommended by others vary from 4 mg/L to 16 mg/L methicillin,22-24 depending on the methods used. And even the breakpoint for oxacillin (i.e., 62 mg/L), recommended by the National Committee for Clinical Laboratory Standards, has been questioned recently because this breakpoint did not correlate well with the presence of met-A gene.25 Further investigation is needed before a definite method for the detection of methicillin resistance can be recommended. We thank Dr Bente Gahm-Hansen, for supplying supported in part by Eli Lilly Denmark A/S.

the specimens

from

Funen

County. This work was

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