Streptococcus pyogenes

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Streptococcus pyogenes
Streptococcus pyogenes
Referentielaboratorium
Het referentielaboratorium voor S. pyogenes is gelokaliseerd in het U.Z.A. - U.I.A. - Antwerpen.
Material and Methods
Strains
A total of 45 Belgian diagnostic laboratories submitted 555 presumptive group A streptococci (GAS) : 3 or more GAS were
received from 22 laboratories; the range of the number of GAS sent per laboratory was 1 to 81 isolates and the mean number
11 isolates.
Among all 555 isolates sent to the reference centre, the identification of S. pyogenes was confirmed for 518 isolates. These
isolates comprised 76 S. pyogenes from sterile sites and 442 organisms from non-sterile sites (including 380 throat swab
samples) (table 7).
Table 7 : S. pyogenes : Source of isolation of 518 GAS
Invasive sites
Non-invasive sites
No
No
Blood
Deep wound
Synovial fluid
Tissue
Cerebrospinal fluid
Pleural fluid
Peritoneal fluid
Other sterile sites
58
3
3
3
2
1
1
5
Throat
Ear
Abcess
Skin
Wound non-sterile
Vagina
Other non-sterile
Unknown
Total
76
Total
380
18
9
10
12
3
5
5
442
For 486 isolates the gender was reported, and 54.3% of the strains were isolated from females. More than 50% of the strains
were collected from patients < 16 years of age (figure 6).
Figure 6 : S. pyogenes : Age distribution of 471 GAS
35
30
number
25
20
15
10
5
88
80
73
64
58
52
47
41
36
31
26
20
15
10
5
0
0
age (years)
Most of the isolates were collected during the periods May-July and October-November (figure 7).
Figure 7 : S. pyogenes : Date of isolation of 485 GAS
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number
50
40
30
20
10
0
1/01/00
1 /03/00
1/05/00
1/07/00
1/09/00
1/11/00
Streptococcus pyogenes
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Streptococcus pyogenes
Referentielaboratorium
Phenotypic detection of resistance mechanisms
Double-disc test using erythromycin (diffusable content 78 µg) and clindamycin (diffusable content 25 µg) Neo-Sensitab discs
(Rosco, Taastrup, Denmark) were placed at 15 to 20 mm apart on a blood agar plate (Mueller-Hinton agar supplemented with
5% defibrinated horse blood) on which a bacterial suspension equivalent to that of a 0.5 McFarland standard had been
previously inoculated. Following overnight incubation at 37°C in aerobic conditions, the inhibition zone around the two discs
determined the inducible, constitutive, or M resistance phenotype of the strain.
Antimicrobial susceptibility testing
MICs were determined by the agar dilution method using the criteria recommended by the National Committee for Clinical
Laboratory Standards. The following drugs were tested: erythromycin, clindamycin, miokamycin, azithromycin, clarithromycin
and tetracycline.
Polymerase chain reaction (PCR)-based detection of the resistance genes
Specific primers were used to detect the genes mefA, ermB (1) and ermTR (2).
Pulsed-field gel electrophoresis
PFGE was performed as described previously (3).
Results
Phenotypic detection of resistance mechanisms
76 (14.7%) of 518 GAS strains tested were erythromycin resistant.
According to the double-disc test, 35 (6.8%) of the erythromycin-resistant isolates showed constitutive resistance (c MLSB)
and 41 (7.9%) exhibited the pattern of novel resistance (M phenotype). Among the invasive isolates the prevalence of c MLSB
and M phenotype was 10.5% and 6.6%, respectively, and among the throat isolates, the prevalence was 6.1% and 8.4%,
respectively.
The prevalence of resistance varied between the laboratories from 0% to 21.4%; the prevalence of ermB and mefA varied
from 0% to 17.1% and from 0% to 12.9%, respectively.
PCR-based detection of the resistance genes
In 95.6% the constitutive resistance phenotype was genotypically confirmed by the presence of the ermB gene, while in 96.9%
the mefA gene was present in the M resistance-phenotype strains. Two strains which were resistant to erythromycin but
negative for ermB and mefA carried the ermTR gene.
Antimicrobial susceptibility testing
The MIC ranges and the MICs at which 50 % and 90% of the 347 throat swab samples are inhibited are given in tables 8 and
9.
Table 8 : S. pyogenes : MIC range, MIC50 , MIC90 (µg/ml) and % resistance for the 347 isolates
Antibiotic
Erythromycin
Clarithromycin
Azithromycin
Miokamycin
Clindamycin
Tetracycline
Range
MIC50
MIC90
No. Resistance (%)
≤ 0.03 - > 512
≤ 0.03 - > 512
≤ 0.03 - > 512
0.0075 - 64
≤ 0.03 - 256
0.03 - 128
0.06
0.03
0.125
0.25
0.03
0.25
16
8
16
0.5
0.125
16
50 (14.4)
49 (14.1)
49 (14.1)
20 (5.8)
19 (5.5)
41 (11.8)
Table 9 : S. pyogenes : Range, MIC50, MIC90 (µg/ml), % resistance and resistance breakpoint for 347 strains of Streptococcus pyogenes for 5
antibiotics
Erythromycin-susceptible strains (297)
Antibiotic
Erythromycin
Clarithromycin
Azithromycin
Miokamycin
Clindamycin
Tetracycline
Erythromycin-resistant strains (50)
Range
MIC50
MIC90
No. Resistance (%)
Resistance
breakpoint
≤ 0.03 - 0.5
≤ 0.03 - 16
≤ 0.03 - 8
0.0075 - 2
≤ 0.03 - 0.5
0.03 - 64
0.03
0.03
0.125
0.25
0.03
0.25
0.06
0.06
0.125
0.5
0.06
0.5
0
2 (0.7)
1 (0.3)
1 (0.3)
0
15 (5.1)
≥1
≥1
≥ 2
≥ 2*
≥1
≥8
Range
2 - >512
0.03 - >512
0.06 - >512
0.125 - 64
≤ 0.03 - >512
0.06 – 128
MIC50
MIC90
16
16
32
0.25
0.06
8
> 512
> 512
512
32
256
64
No. Resistance
(%)
50 (100)
47 (94.0)
48 (96.0)
19 (38.0)
19 (38.0)
26 (52.0)
* No defined NCCLS breakpoint
50 (14.4%) of the throat swab samples isolates tested were resistant to erythromycin, 5.5% of the isolates were resistant to
clindamycin. The resistance to clarithromycin and azithromycin was 14.1%, whereas the resistance to miokamycin was 5.8%.
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Streptococcus pyogenes
Referentielaboratorium
Streptococcus pyogenes
The erythromycin-resistant isolates, irrespective of the phenotype, were resistant to the 14- en 15- membered macrolides,
clarithromycin and azithromycin in respectively 94.0% en 96.0% of the isolates. In contrast, clindamycin and miokamycin
retained some activity, depending on the phenotype; all of the novel resistance strains, but non of the constitutive resistance
strains were susceptible to these agents.
Pulsed-field gel electrophoresis
All throat swab samples strains were typed by PFGE with the resitriction enzyme SmaI or SfiI depending on the phenotype.
Visual and computerised analysis obtained 21 unrelated GAS genotypes. One frequently found genotype comprise 25.5% of
the isolates. The 3 most frequently found genotypes comprise 49.1% of the isolates studied.
References
1.
Sutcliffe, J., A. Tait-Kamradt, and L. Wondrack. 1996. Streptococcus pneumoniae andStreptococcus pyogenes resistant
to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system. Antimicrob.
Agents Chem. 40: 1817-1824.
2.
Seppälä, H., M. Skurnik, H. Soini, M. Roberts, and P. Huovinen. 1998. A novel erythromycin resistance methylase gene
(ermTR) in Streptococcus pyogenes. Antimicrob. Agents Chem. 42: 257-262.
3.
Descheemaeker, P., S. Chapelle, C. Lammens, M. Hauchecorne, M. Wijdooghe, P.Vandamme, M. Ieven, and H.
Goossens. 2000. Macrolide resistance and erythromycin resistance determinants among Belgian Streptococci pyogenes
and Streptococci pneumoniae isolates. J. Antimicrob. Chemother. 45:167-173.
Streptococcus pyogenes
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