Susceptibility profile to penicillin, erythromycin and clindamycin of clinical isolates of group B streptococci recently isolated in Belgium and detection of erythromycin resistance genes

(1)

pm-chulg - 16.11.07 1 P.Melin

P.Melin, , C.MegaliC.Megali, , MP.HayetteMP.Hayette, P.De Mol, P.De Mol

Medical

Medical microbiologymicrobiology, , UniversityUniversity hospitalhospitalof Liegeof Liege Belgian

Belgian referencereference laboratorylaboratory for GBSfor GBS

Susceptibility profile to penicillin,

erythromycin and

clindamycin

of clinical

isolates of group B streptococci recently

isolated in Belgium and detection of

erythromycin resistance genes

Abstract C2

Abstract C2-168-168

47th ICAAC, Chicago,

(2)

Background

Group B

streptococci

or

S.agalactiae

Since

the

1970s,

leading

cause of

life

-

-theatening

theatening

infections in

newborns

Neonatal

illness

/

death

Long-

Long

-term

term

disabilities

Maternal

morbidity

Along

pregnancy

Peripartum

Serious

diseases

among

elderly

and

adults

with

underlying

diseases

Significant

mortality

(3)

(4)

Prevention

of

_of

perinatal

_perinatal

GBS EOD

_{GBS EOD}

Intrapartum

antibiotics

Highly

effective

at

preventing

EOD

in

women

at

risk

of

transmitting

GBS

to

their

newborns

(

>

4 h)

INTRAPARTUM ANTIMICROBIAL

PROPHYLAXIS

Main goal

:

To

prevent

70 to 80 %

of

GBS EO cases

Secondary

:

(5)

(6)

Concerns

Increase

of

resistance

to

erythromycin

and

clindamycin

Susceptibility

to

penicillin

Very

few

R

isolates

recently

characterized

in

Japan

(7)

pm-chulg - 16.11.07 7

Objectives

Among

GBS

recently

isolated

in

Belgium

From

adults

with

severe

infections

From

early

or

late

onset

neonatal

diseases

To monitor

penicillin

susceptibility

To

determine

rates of

erythomycin

and

clindamycin

resistance

To

assess

the

distribution of macrolide

resistance

phenotypes

To

identify

genes

coding

for

resistance

to

erythromycin

(8)

Methods

:

_:

Isolates

_Isolates

Clinical

isolates

Sent to Sent to thethe BelgianBelgian referencereference laboratorylaboratory for GBS from for GBS from JanuaryJanuary 2005

2005 andand JuneJune 20062006

FromFrom bloodblood, CSF or , CSF or anyany deepdeep normallynormally sterilesterile sitesite

178 178 isolatesisolates

22 from 22 from neonatalneonatal EODEOD

10 from 10 from neonatalneonatal LODLOD

146 from 146 from adultadult invasive invasive diseasedisease

SerotypesSerotypes

IaIa (17%); Ib(17%); Ib (6%); II (10%); III (33%); IV (6%); V (21%); others(6%); II (10%); III (33%); IV (6%); V (21%); others (7%)(7%)

Reference

strains

Positive Positive eithereither for for ermermBB, , ermermTRTR or or mefmefAA genesgenes

(9)

pm-chulg - 16.11.07 9

Methods

:

_:

Susceptibility

testing

Disk

diffusion

For all For all isolatesisolates

ErythromycinErythromycin (15 (15 µµg) g) andand clindamycinclindamycin (2 (2 µµg) g) disksdisks

18 mm 18 mm apartapart

Etest

BenzylpenicillineBenzylpenicilline stripsstrips

For all For all isolatesisolates

ErythromycinErythromycin andand clindamycinclindamycin stripsstrips

For all For all erythromycinerythromycin resistantresistant isolatesisolates

Macrolide

resistant

phenotypes

-

Dtest

MLSMLS_B_Bphenotypesphenotypes

InducibleInducible ResistanceResistance

Constitutive Constitutive ResistanceResistance

(10)

Interpretation

criteria

_criteria

(MH

with

blood

)

(CLSI 2006)

Zone

Zone MIC MIC

Diameter Diameter (mm) (mm) (mg/L)(mg/L) S S I I R R S S I I RR

Penicillin

>> 2424 -- -- < < 0.120.12 -- -

-Erythromycin

Erythromycin

>> 2121 1616--2020 << 1515 < < 0.250.25 0.50.5 >> 11

Clindamycin

>> 1919 1616--1818 << 1515 < < 0.250.25 0.50.5 >> 11

(11)

pm-chulg - 16.11.07 11

Methods

:

_:

Detection

of R

genes

DNA extraction

QIAmpQIAmp DNA Mini Kit (DNA Mini Kit (QiagenQiagen))

PCR amplification

with

specific

primers

and

protocols

DetectionDetection of of ermermBB, , ermermTRTR andand mefmefAA genesgenes

Caracterisation

of PCR

products

SeparationSeparation by by electrophoresiselectrophoresis

2% Agarose gel + 2% Agarose gel + ethidiumethidium bromidebromidestainingstaining

VisualizationVisualization underunder UV lightUV light

640 640 530 530 348 348 erm ermBB erm ermTRTR mef mefAA PCR

PCR ProductProduct sizessizes(bp(bp)) Targets

(12)

Results

Antimicrobial

susceptibility

profile of

178 GBS

clinical

isolates

% of Resistance

MICMIC₉₀₉₀ MIC Range MIC Range

(mg/L) (mg/L) (mg/L)(mg/L)

Penicillin*

0

0.094

0.016

0.016 -

-

0.094

0.094 Erythromycin

Erythromycin

32 ₃₂

Clindamycin

19

(25**)(25**)

* : same interpretation for ampicillin and cefazolin **: iMLS resistant phenotype included

(13)

pm-chulg - 16.11.07 13

Results

Erythromycin

and

clindamycin

resistance

Evolution

among

Belgian

GBS

isolates

0 5 10 15 20 25 30 35 1990 1999 2001-2003 2005-2006 % o f R Erythromycin Clindamycin

(14)

Results

Erythromycin

resistance

_resistance

among

_among

Belgian

clinical

GBS

isolates

0 10 20 30 40 50 60 Neonatal infection Adult infection Ia Ib II III IV V Others % o f R e s is ta n c e 2001-03 2005-06

(15)

pm-chulg - 16.11.07 15

Results

MLS

Resistance

phenotypes

Dtest

cMLS Erythro R & Clinda R

iMLS Erythro R & Clinda S/I/R with Dtest +

M Erythro R & Clinda S with Dtest -4 / 12 4 / 12 21 21 M M 4 / >256 4 / >256 34 34 Inducible Inducible >256 / >256 >256 / >256 45 45 Constitutive Constitutive MLS MLS Ery

Ery MICMIC₅₀₅₀/ / MICMIC₉₉₀₀

( (mg/L)mg/L) % % Phenotype Phenotype

(16)

Results

Distribution of macrolide R

genes

_genes

3 (25)

8 (67)

1 (8)

ermTR

mefA

ermB & ermTR

M

(12 isolates) 1 (5) 16 (80) 1 (5) 1 (5) 1 (5) ermB ermTR

ermB & ermTR ermTR & mefA

unknown

MLS inducible

(20 isolates) 19 (73) 2 (8) 5 (19) ermB ermTR

ermB & ermTR

MLS constitutive

(

26 isolates)

Number

of

isolates

(%

per

phenotype

)

Resistance

genotype

Resistance

phenotype

(17)

pm-chulg - 16.11.07 17

Conclusion

All GBS isolates fully susceptible to penicillin

Increase of resistance to

macrolides

: a relevant

problem

.

Level: similar to rates observed in France, a neighbour country.

No more difference among isolates from either adults or neonates.

Most of macrolide R isolates had a MLS phenotype.

Detection of MLS-IR is important

simple and reliable double-disk diffusion test strongly recommended.

Neither macrolides no lincosamides should no longer

be used without susceptibility testing

.

(18)

C.Megali C.Megali MP.Hayette MP.Hayette P.De Mol P.De Mol Technical

Technical staff of staff of thethe medicalmedical microbiology

microbiology laboratorylaboratory of of thethe University

University hospitalhospital of Liof Lièègege Laboratories

Laboratories of of thethe BelgianBelgian surveillance network