Folia Microbiol DOI 10.1007/s12223-011-0017-1
Antibiotic resistance and molecular characterization of clinical isolates of methicillin-resistant coagulase-negative staphylococci isolated from bacteremic patients in oncohematology O. Bouchami & W. Achour & M. A. Mekni & J. Rolo & A. Ben Hassen
Received: 22 September 2010 / Accepted: 18 January 2011 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2011
Abstract Polymerase chain reaction (PCR) amplification of antibiotic resistance genes as well as staphylococcal cassette chromosome mec (SCCmec) typing and pulsedfield gel electrophoresis (PFGE) of SmaI macrorestriction fragments of genomic DNA were used to characterize 45 methicillin-resistant coagulase-negative staphylococci (MRCoNS) isolates responsible of bacteremia recovered in patients at the Bone Marrow Transplant Centre of Tunisia in 1998–2007. Among the 45 MRCoNS isolates, Staphylococcus epidermidis was the most prevalent species (75.6%) followed by Staphylococcus haemolyticus (22.2%) and Staphylococcus hominis (2.2%). Extended susceptibility profiles were generated for MRCoNS against 16 antimicrobial agents. Out of 45 mecA-positive strains, 43 (95.6%) were phenotypically methicillin-resistant and two (4.4%) were methicillin-susceptible. The msr(A) was the most prevalent gene (13 isolates; 48.1%) among erythromycin-resistant isolates. The erm(C) was found alone in seven (25.9%) or in combination with both erm (A) and erm(B) in two (7.4%) isolates. The aac(6′)-Ie-aph O. Bouchami : W. Achour : M. A. Mekni Laboratory Department, Centre National de Greffe de Moelle Osseuse, Tunis, Tunisia J. Rolo : A. B. Hassen Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB/UNL), Oeiras, Portugal O. Bouchami (*) Service des laboratoires, Centre National de Greffe de Moelle Osseuse, Rue Djebel Lakhdhar, Bab Saadoun, 1006 Tunis, Tunisia e-mail:
[email protected]
(2″)-Ia was the most prevalent gene among aminoglycosideresistant isolates, detected alone in 14 isolates (33.3%) isolates, in combination with ant(4′)-Ia in 18 (42.8%) isolates, in combination with aph(3′)-IIIa in four (9.5%) or with both ant(4′)-Ia and aph(3′)-IIIa in two (4.7%) isolates. The ant(4′)Ia was detected in three (7.1%) isolates and the aph(3′)-IIIa in one (2.4%) isolate. Among tetracycline-resistant isolates, six (85.7%) strains harbored the tet(K) gene and one (14.3%) strain carried tet(K) and tet(M) genes. SCCmec types IV (31%) and III (24.5%), the most prevalent types detected, were found to be more resistant to non-β-lactam antibiotics. A wide diversity of isolates was observed by PFGE among MRCoNS. Abbreviations CoNS Coagulase-negative staphylococci CVC Central venous catheter MLSB Resistance to macrolides, lincosamides, and streptogramin B (other than erythromycin) MRCoNS Methicillin-resistant coagulase-negative staphylococci PCR Polymerase chain reaction PFGE Pulsed-field gel electrophoresis SCCmec Staphylococcal cassette chromosome mec
Coagulase-negative staphylococci (CoNS) are the largest cause of bloodstream and central venous catheter (CVC)related bloodstream infections among patients with hematological disorders. To a large extent, this results from their ability to accumulate antibiotic resistance determinants (Worth and Slavin 2009). Methicillin-resistant staphylococcal strains have acquired and integrated into their genome the staphylococcal cassette chromosome mec (SCCmec), which
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carries the methicillin resistance (mecA) gene, and other antibiotic resistance determinants. SCCmec consists of the mec gene and cassette chromosome recombinase (ccr) gene complex (Ito et al. 2004). To date, eight types of SCCmec have been found in S. aureus and in other staphylococcal species (IWG-SCC 2009). In staphylococci, the most frequent aminoglycoside-modifying enzymes is the bifunctional enzyme AAC(6′)/APH(2″) encoded by the gene aac (6′)-Ie-aph(2″)-Ia. It modifies all clinically available aminoglycosides, except streptomycin (Shaw et al. 1993). The APH(3′)-III enzyme encoded by aph(3′)-IIIa gene mediates essentially resistance to kanamycin. Whereas, the ANT(4′)-I enzyme encoded by ant(4′)-Ia gene mediates resistance to both kanamycin and tobramycin (Ounissi et al. 1990). Streptomycin resistance is mediated by mutation in chromosomal genes encoding ribosomal proteins or by production of the ANT(6)-I enzyme encoded by the ant(6)-Ia gene (Shaw et al. 1993). Erythromycin resistance in CoNS is associated most often with the presence of an RNA methylase, whose action also affects resistance to other macrolides, lincosamides, and streptogramin B (MLSB). This resistance is mediated by the erm-type genes, caused almost exclusively by erm(A) or erm(C). The structural genes may be expressed either inducibly or constitutively. A second resistance mechanism involves export of the macrolide antibiotics, typically mediated by mrs(A) (Gatermann et al. 2007). Two known mechanisms of tetracycline resistance have been found in CoNS. In one, a membrane protein, encoded by tet(K) or tet(L) genes, mediates active efflux of the drug and in the second a cytoplasmic protein, encoded by tet(M) or tet(O) genes, reduces the sensitivity of the ribosome to the drug (Chopra and Robert 2001). Multiple DNA-based methods have been introduced to type CoNS strains genetically. Pulsed-field gel electrophoresis (PFGE) is the method of choice for local epidemiological studies of these species. Complete characterization of CoNS also requires identification of the structural types SCCmec element. SCCmec typing of CoNS may serve as a useful tool for clinicians and epidemiologists in their effort to prevent and control infections caused by these organisms. In this work, we analyze the distribution of resistance genes for various antibiotics in a collection of methicillinresistant coagulase-negative staphylococci (MRCoNS) clinical strains responsible of bacteremia, isolated in a 10-year period from hospitalized patients. In addition, molecular characterization using PFGE in combination with SCCmec typing was realized.
Materials and methods Bacterial isolates and growth conditions From 1998 to 2007, 45 MRCoNS clinical isolates responsible for bacter-
emia (each strain was isolated in two independent positive blood cultures or in positive peripherical blood and CVC cultures for identical isolates of CoNS) included 34 Staphylococcus epidermidis, ten Staphylococcus haemolyticus, and one Staphylococcus hominis, were collected in the Bone Marrow Transplant Centre of Tunisia and were obtained from blood cultures (91%) and from CVC (9%) of neutropenic patients. The samples were initially identified by colony morphology on Mueller–Hinton (MH) agar plates, mannitol fermentation, Gram characteristics, catalase, coagulase tests, and DNAase activity. Isolates were characterized at the species level by API ID32 Staph system (bioMérieux, France) according to the instructions of the manufacturer. The organisms were stored in MH broth with 10% sterile glycerol at −20°C. Antibiotic susceptibility testing Susceptibility tests were performed by the disk diffusion method on the MH agar plates (Difco) with commercial antibiotic disks (Sanofi Diagnostics, Pasteur). Erythromycin, clindamycin, and pristinamycin were tested in separate plates by the disk diffusion method to differentiate the MLSB resistance phenotype as constitutive, inducible, or M phenotype. Minimum inhibitory concentrations (MICs) for oxacillin (Biochemie Gmlott, Australia), erythromycin (Abbott, France), gentamicin (A. Menarini, Italy), streptomycin and tetracycline (Sigma-Aldrich, France), vancomycin (Eli-Lilly France, France), and teicoplanin (Aventis) were determined by agar dilution method. Results were interpreted according to the guidelines of the Comité de l’Antibiogramme de la Société Française de Microbiologie (http://www.sfm.asso.fr). S. aureus ATCC25923 was used as a quality control strain. PCR amplification Genomic DNA was extracted from staphylococcal cultures and used as a template for amplification. The presence of mecA was tested according to the polymerase chain reaction (PCR) assay according to Frebourg et al. (2002). Primers for mecA (Forward 5′GTA GAA ATG ACT GAA CGT CCG ATA A-3′ and reverse 5′-CCA ATT CCA CAT TGT TTC GGT CTA A-3′) containing a ClaI restriction site were designed by us from published GenBank sequences (accession no. NC002952) to provide a PCR product of 683 bp. erm(A), erm(B), erm (C) were determined by multiplex and msr(A) and mef(A) by duplex PCRs (Martineau et al. 2000). Oligonucleotide primers and conditions for aac(6′)-Ie-aph(2″)-Ia, aph(3′)IIIa, ant(6)-Ia, ant(4′)-Ia were published by Kobayashi et al. (2001) and those of tet(K), tet(L), tet(M), and tet(O) genes were described by Trzcinski et al. (2000). All PCR amplifications were performed in a Perkin Elmer 4600 (USA), and the products were analyzed by electrophoresis on 1.5% agarose gels.
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The following control strains were included: S. epidermidis WHO36 (mecA) (World Health Organization), Staphylococcus aureus S5 (mrsA), S. aureus HM1051 (ermA, ermC), S. aureus HM1054/R (ermC), Streptococcus pneumoniae HM28 (ermB), S. pneumoniae O2J1157 (mefA), S. pneumonia P9 (ermB, mefA), Enterococcus faecalis E9 (aac (6′)-Ie-aph(2″)-Ia) (Angot et al. 2000; Pavie et al. 2002) (kindly provided by Prof. R. Leclercq, Department of Microbiology, CHU, Côte de Nacre, Caen, France) and S. aureus N315 (ant(4′)-Ia, mecA) (kindly provided by Prof. T. Ito, Department of Bacteriology, Faculty of Medicine, Jutendo University, Tokyo, Japan) (Ito et al. 2004). SCCmec type assignment Three multiplex SCCmec typing PCRs were performed with template DNA from each isolate to determine if they harbored segments of SCCmec elements I to V according to Zhang et al. (2005) and Ito et al. (2001). The first multiplex PCR detects the presence of SCCmec types and subtypes I, II, III, IVa, IVb, IVc, IVd, and V and the presence of mecA gene as an internal control. The two other multiplex PCR assays detects the presence of class A and class B mec gene (Zhang et al. 2005) and the presence of the ccr complex genes ccrAB1, ccrAB2, and ccrAB3 (Ito et al. 2001). Class C2 and ccrC were detected by simplex PCR (Zhang et al. 2005; Okuma et al. 2002). SCCmec was considered as non-typeable when either ccr or the mec complex or both were non-typeable. The following S. aureus control strains were used as positive controls for SCCmec typing PCR as indicated: NCTC10442 (SCCmec I, class B mec, ccrAB1); N315 (SCCmec II, class A mec, ccrAB2); 85/2082 (SCCmec III, class A mec, ccrAB3); JCSC 4744 (SCCmec IVa, class B mec, ccrAB2); and WIS (SCCmec V) (Ito et al. 2004). PFGE Agarose disks containing chromosomal DNA were prepared and PFGE was done according to Chung et al. (2000). Genomic DNA was digested in situ with SmaI (Promega). Macrorestriction fragments were separated using a Bio-Rad CHEF DR III apparatus according to Chung et al. (2000) with the exception of the running time, which was set to 22 h. S. epidermidis RP62A (ATCC 35984) was used to access inter-gel reproducibility of mobility; λ ladder PFGE marker was used as a molecular size standard. After electrophoresis, the DNA was stained using ethidium bromide, visualized and photographed under ultraviolet light. PFGE profiles obtained were analyzed with BioNumerics Software (version 4.5) from Applied Maths (Belgium). Clustering was performed using the Dice similarity coefficient and the unweighted pair group method with arithmetic means, with 1.3% of tolerance and 0.8% optimization (Miragaia et al. 2008). PFGE types were automatically assigned by using cutoff similarity value of 79%. The later analysis was confirmed
by visual inspection according to the criteria of Tenover et al. (1995), and the validated profiles were directly compared.
Results Antimicrobial susceptibility Methicillin resistance was detected in 43 of the 45 strains studied (95.6%) (MIC90 >256 μg/mL): 32 S. epidermidis, ten S. haemolyticus, and one S. hominis. Erythromycin resistance was found in 27 strains (60%) (MIC90 >256 μg/mL), of which 12 (44.5%) showed a constitutive MLSB phenotype, two (7.4%) an inducible MLSB phenotype, and 13 (48.1%) had M phenotype. Overall, 42 (93.3%) were resistant to kanamycin, 40 (88.9%) to tobramycin, 35 (77.8%) to gentamicin (MIC90 >1024 μg/mL), and nine (18.4%) to streptomycin (MIC90 >1,024 μg/mL). Tetracycline resistance was observed in seven (15.6%) isolates (MIC90 =48 μg/mL). Resistance was also observed in 24 (53.3%) strains to rifampin, in 32 (71.1%) strains to cotrimoxazole, in 30 (66.7%) strains to ciprofloxacin, in 21 (46.7%) strains to fosfomycin, in 28 (62.2%) strains to fusidic acid, and in five (11%) strains to chloramphenicol. Only two (4.4%) strains exhibited intermediate susceptibility to teicoplanin (MIC values were between 8 and 16 μg/mL), and no isolate was resistant to vancomycin or pristinamycin. Prevalence of resistance genes The mecA was detected in the 45 CoNS strains: 43 strains were methicillin-resistant, according to antimicrobial susceptibility tests, and two were methicillin-susceptible S. epidermidis strains. The msr(A) was the most prevalent gene (13 isolates; 48.1%) among erythromycin-resistant isolates. The erm(C) was found alone in seven (25.9%) or in combination with both erm (A) and erm(B) in two (7.4%) isolates. The aac(6′)- Ie-aph (2″)-Ia was the most prevalent gene among aminoglycoside-resistant isolates, detected alone in 14 isolates (33.3%) isolates, in combination with ant(4′)-Ia in 18 (42.8%) isolates, in combination with aph(3′)-IIIa in four (9.5%) or with both ant(4′)-Ia and aph(3′)-IIIa in two (4.7%) isolates. The ant(4′)-Ia was detected in three (7.1%) isolates and the aph(3′)-IIIa in one (2.4%) isolate. Among tetracycline-resistant isolates, six (85.7%) strains harbored the tet(K) gene and one (14.3%) strain carried tet(K) and tet (M) genes. SCCmec typing All 45 mecA-positive CoNS strains were studied for SCCmec types. Twenty-eight (62%) strains were typeable and belonged to S. epidermidis species. The most prevalent SCCmec type was SCCmec IV (31%), followed by SCCmec III (24.5%), SCCmec II (4.5%) and SCCmec V (2%) (Table 1). A total of 17 out of the 45 (38%) strains
Folia Microbiol Table 1 Phenotypic and genotypic characteristics of the MRCoNS Strain
Species
Year of isolation
Ward
Clinical product
Resistance pattern
S1
SE
2006
HU
BL
P, Ox, G, K, T, E, C, Ch, Rf, Fu
S2
SE
2006
HU
BL
P, Ox, G, K, T, E, C, Rf, Cp, Fu
S3
SE
2005
GU
CVC
S4
SE
2005
GU
BL
S5
SE
2006
HU
BL
S6 S7
SE SE
1999 2007
GU HU
BL BL
S8
SE
2007
HU
BL
P, Ox, G, K, T, E, Rf, SXT, Cp, Fo, Fu P, Ox, G, K, T, E, C, Rf, SXT, Cp, Fo, Fu P, Ox, G, K, T, E, C, Tc, SXT, Cp, Fu P, Ox, Ch, Rf, Te P, Ox, K, T, C, Tc, Rf, Te P, Ox, K, T, Tc, Rf
S9
SE
2006
HU
BL
S10
SE
2007
GU
BL
S11
SE
2005
GU
BL
S12
SE
2007
GU
CVC
S13
SE
2007
IHU
BL
S14
SE
2005
HU
BL
S15
SE
2006
GU
BL
S16
SE
2004
GU
BL
S17
SE
2005
GU
BL
S18 S19
SE SE
2005 2005
HU GU
BL BL
S20 S21
SE SE
2004 1999
GU GU
BL CVC
P, Ox, S, K, SXT P, Ox, K, C, E, Rf, SXT, Fo
S22
SE
2006
GU
BL
S23
SE
2007
IHU
BL
S24
SE
2006
GU
BL
P, Ox, G, K, T, E, C, Rf, SXT, Cp, Fo, Fu P, Ox, G, K, T, Tc, Rf, SXT, Cp, Fo, Fu P, Ox, S, G, K, T, Ch, SXT, Cp, Fu
P, Ox, G, K, T, E, Fo P, Ox, S, G, K, T, E, C, SXT, Cp, Fo, Fu P, Ox, K, T, Tc, SXT, Fo, Fu P, Ox, S, G, K, T, Tc, Rf, SXT, Cp, Fo, Fu P, Ox, G, K, T P, Ox, G, K, T, SXT, Fo P, Ox, G, K, T, SXT, Cp, Fo P, OX, G, K, T, E
P, Ox, G, K, T, E, C, Rf, SXT, Cp, Fu P, Ox, E, Fu P, Ox, G, K, T, E, Fo, Fu
Gene content
SCCmec typing
PFGE type
mec complex
ccr type
SCCmec
erm(A), erm(B), aac (6′)-Ie-aph(2″)-Ia, ant (4′)-Ia, mecA erm(A), erm(B), aac (6′)-Ie-aph(2″)-Ia, ant (4′)-Ia, mecA erm(A), erm(B), erm(C), aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, mecA erm(A), erm(B), erm(C), aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, mecA erm(C), aac(6′)-Ie-aph (2″)-Ia, aph(3′)-IIIa, tet(K), mecA mecA ant(4′)-Ia, tet(M), tet(K)
class B
ccrAB2
IV
J
class B
ccrAB2
IV
K
class B
ccrAB2
IV
E1
class B
ccrAB2
IV
E2
class B
ccrAB2
IV
F1
NT class A
ccrAB2 ccrAB3
NT III
F2 G1
ant(4′)-Ia, tet(K), mecA
class A
NT
NT
G2
msr(A), aac(6′)-Ie-aph (2″)-Ia, mecA erm(A), aac(6′)-Ie-aph (2″)-Ia, mecA
class B
ccrAB2
IV
B1
class A
ccrAB3
III
B2
aac(6′)-Ie-aph(2″)-Ia, tet(K), mecA aac(6′)-Ie-aph(2″)-Ia, tet(K), mecA
class B
ccrAB2
IV
B3
class B
ccrAB2
IV
B4
aac(6′)-Ie-aph(2″)-Ia, mecA aac(6′)-Ie-aph(2″)-Ia, mecA aac(6′)-Ie-aph(2″)-Ia, mecA msr(A), aac(6′)-Ie-aph (2″)-Ia, aph(3′)-IIIa, mecA erm(A), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, mecA msr(A), mecA msr(A), aac(6′)-Ie-aph (2″)-Ia, aph(3′)-IIIa, mecA aph(3′)-IIIa, mecA erm(C), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, aph (3′)-IIIa, mecA erm(C), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, mecA aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, tet(K), mecA aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, mecA
class B
ccrAB2
IV
B5
class B
ccrAB2
IV
C1
class B
ccrAB2
IV
C2
class B
ccrAB2
IV
C3
class B
ccrAB2
IV
C4
class A class A
ccrAB2 NT
II NT
H1 H2
class B class A
ccrAB2 ccrAB2
IV II
I1 I2
class A
ccrAB3
III
A1
class A
ccrAB3
III
A2
class A
ccrAB3
III
A3
Folia Microbiol Table 1 (continued) Strain
Species
Year of isolation
Ward
Clinical product
Resistance pattern
S25
SE
2007
HU
BL
P, Ox, G, K, T, E, C, SXT, Cp, Fo, Fu P, Ox, G, K, T, Rf, SXT, Cp, Fo, Fu P, Ox, G, K, T, Ch, SXT, Cp, Fu P, Ox, G, K, T, E, C, Ch, Rf, SXT, Cp, Fo, Fu P, Ox, G, K, T, Rf, SXt, Cp, Fo, Fu P, Ox, G, K, T, E, C, Rf, SXT, Cp, Fo, Fu P, G, K, T, Rf, SXT, Cp, Fo, Fu P P, Ox, K, T, E, Tc, Rf, SXT, Cp, Fo, Fu P, Ox, G, K, T, Rf, SXT, Cp, Fu P, Ox, S, G, K, T, E, SXT, Cp
S26
SE
1998
HU
BL
S27
SE
2006
GU
BL
S28
SE
2005
GU
BL
S29
SE
2005
GU
BL
S30
SE
2005
GU
BL
S31
SE
2000
GU
CVC
S32 S33
SE SE
2005 2007
GU HU
BL BL
S34
SE
2006
GU
BL
S35
SHae
2007
HU
BL
S36
SHae
2003
HU
BL
P, Ox, S, G, K, T, E, Rf, SXT, Cp
S37
SHae
2006
HU
BL
P, Ox, S, G, K, T, E, SXT, Cp
S38
SHae
2005
HU
BL
S39
SHae
2006
GU
BL
S40
SHae
2005
HU
BL
S41 S42
SHae SHae
2007 2006
HU GU
BL BL
S43
SHae
2004
GU
BL
P, Ox, G, K, T, E, Rf, SXT, Cp, Fu P, Ox, S, G, K, T, E, Rf, SXT, Cp, Fo, Fu P, Ox, S, G, K, T, E, C, Rf, SXT, Cp, Fo P, Ox, K, T, E, Fu P, Ox, S, G, K, T, SXT, Cp P, Ox, G, K, T, E, SXT, Cp
S44
SHae
2007
IHU
BL
S45
SHo
2005
GU
BL
P, Ox, G, K, T, E, Ch, Rf, Cp, Fu P, Ox, G, K, T, E, SXT, Cp, Fu
Gene content
SCCmec typing
PFGE type
mec complex
ccr type
SCCmec
erm(C), aac(6′)-Ie-aph (2″)-Ia, mecA
class A
ccrAB3
III
A4
aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, mecA aac(6′)-Ie-aph(2″)-Ia, mecA erm(C), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, mecA aac(6′)-Ie-aph(2″)-Ia, mecA erm(C), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, mecA aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, mecA mecA msr(A), ant(4′)-Ia, tet (K), mecA
class A
ccrAB3
III
A5
class A
ccrAB3
III
A6
class A
ccrAB3
III
D1
class A
New2
D2
class A
ccrAB2 +3 ccrAB3
III
D3
NT
NT
NT
D4
NT class C2
NT ccrC
NT V
L M
aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, mecA msr(A), aac(6′)-Ie-aph (2″)-Ia, aph(3′)-IIIa, mecA msr(A), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, aph (3′)-IIIa, mecA msr(A), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, mecA msr(A), aac(6′)-Ie-aph (2″)-Ia, mecA msr(A), aac(6′)-Ie-aph (2″)-Ia, mecA
class A
ccrAB3
III
N
NT
NT
NT
C'
NT
ccrAB2
NT
D'
NT
NT
NT
B1´
NT
NT
NT
B2´
NT
NT
NT
B3´
class A
ccrAB1
New1
E´
class A NT
NT NT
NT NT
A1´ A2´
NT
NT
NT
A3´
NT
NT
NT
F´
class A
ccrAB1
New1
–
erm(C), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, mecA msr(A), ant(4′)-Ia, mecA aac(6′)-Ie-aph(2″)-Ia, mecA msr(A), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, mecA msr(A), aac(6′)-Ie-aph (2″)-Ia, mecA erm(C), aac(6′)-Ie-aph (2″)-Ia, ant(4′)-Ia, mecA
SE S. epidermidis, SHae S. haemolyticus, SHo S. hominis, HU hematological unit, IHU immunohaematological unit, GU graft unit, BL blood, CVC central venous catheter, P penicillin G, Ox oxacillin, S streptomycin, G gentamicin, K kanamycin, T tobramycin, E erythromycin, C clindamycin, Tc tetracycline, Ch chloramphenicol, Rf rifampin, SXT cotrimoxazole, Cp ciprofloxacin, Te teicoplanin, Fo fosfomycin, Fu fusidic acid, NT non-typeable, New1 1A, New2 2+3A
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examined showed SCCmec structures that remained nontypeable for a variety of reasons. Two strains showed nontypeable ccr but known mec complex structures and the reverse was true in the case of two strains. New combinations between mec and ccr complexes were observed in three strains. In ten strains, neither ccr nor mec complex were typeable. Isolates with SCCmec types III and IV were found to be more resistant to non-β-lactam antibiotics and harboring combination of the resistance genes tested. Genes aac(6′)-Ie-aph(2″-Ia), ant(4′)-Ia, and erm(C) were the most encountered genes in SCCmec type III (10/11, 8/11, and 4/11, respectively). Whereas, aac(6′)Ie-aph(2″)-Ia, ant(4′)-Ia and erm(A) were the most prevalent in SCCmec type IV (13/14, 5/14, and 5/14, respectively). SCCmec types in S. epidermidis appear to have occurred over the years. Indeed, the majority of isolates of SCCmec type III were collected in 2006 and 2007 and the majority of isolates of SCCmec type IV were collected in 2005 and 2006. PFGE types Macrorestriction fragment analysis of 34 mecApositive S. epidermidis isolates and ten mecA-positive S. haemolyticus showed that they were clustered above 79% similarity in 14 (A to N) and six (A´ to F´) pulsotypes, respectively. It is noteworthy that SCCmec type IV was distributed among seven clusters (B, C, E, F, I, J, K), SCCmec type III among five clusters (A, B, D, G, N) and SCCmec type II in two pulsotypes (H and I) during several years. These data suggest that there is, probably, transfer of SCCmec among S. epidermidis strains and that certain clones are both preferential recipients for specific SCCmec types (SCCmec IV and III) and more successful at environmental competition, having survived for many years (Table 1). Besides, most of the strains with PFGE types B and C carried SCCmec type IV and strains with PFGE type A carried SCCmec type III. Nevertheless, this association between genetic background and SCCmec type was not exclusive.
Discussion The predominance of S. epidermidis among the CoNS (75.6%) was in keeping with previous reports (Akpaka et al. 2006; Abbassi et al. 2008). Bloodstream infection with S. epidermidis, and less commonly with S. haemolyticus, usually involves implantation of medical devices (Akpaka et al. 2006). Results of antibiotic susceptibility testing showed multidrug resistance and variability in resistance patterns, similar to the study of Mohan et al. (2002). In our study, very high antimicrobial resistance rates were observed and 95.5% of the isolates were resistant to more than four
antibiotics. A high rate of methicillin resistance (95.6%) was confirmed in the clinical isolates belonging to three species. This rate is in agreement with other previous reports done in other countries, such as Turkey (74.4%) and France (71%) (Sader et al. 2007; Koksal et al. 2007). MRCoNS demonstrated high rates of resistance to multiple antimicrobial agents in Europe (Stefani and Varaldo 2002) and in Tunisia (Ben Jemaa et al. 2004; Abbassi et al. 2008). It is important to note that no vancomycin resistance was found in the present study and prudent use of vancomycin should be maintained. However, two isolates exhibited intermediate susceptibility to teicoplanin (MIC=8–16 μg/mL). Reduced susceptibility to teicoplanin has been reported in hospital where glycopeptides are extensively used in the empirical treatment of febrile patients with neutropenia (Erjavec et al. 2000). The heavy use of antibiotics, including vancomycin, in certain hospital facilities may select for multiple-resistant commensal organisms, such as methicillin-resistant S. epidermidis (Wu et al. 2001). All except two mecA-positive CoNS isolates held more than one antimicrobial resistance gene and 12 (26.7%) carried four different genes. In particular, two S. epidermidis isolates carried six antimicrobial resistance genes that confer resistance to four different antibiotics. These results confirm the large spread of multidrug-resistant CoNS isolated from clinical samples (Santos Sanches et al. 2000). The fact that 90.5% of our isolates carried the aac (6′)-Ie-aph(2″)-Ia gene reveals a great diffusion of aminoglycoside resistance (Klingenberg et al. 2004). This gene is often carried by conjugative transposons and is usually more diffused in staphylococci (Werckenthin et al. 2001). A large number (48.1%) of the erythromycin-resistant strains harbored msr(A) gene, followed by the erm(C) gene (25.9%). The msr(A) is widespread in CoNS more than in S. aureus (Lina et al. 1999), it is located on large plasmids and may be associated with the erm(C) (Barrière et al. 1998). This could explain the diffusion of these two genes in our collection. Conversely, the erm(A) and erm(B) genes were detected in a lower number of isolates. The tet(K) and tet(M) genes for tetracycline resistance were detected in 85.7% and 14.3%, respectively. In fact, the presence of tet (K) gene on small multicopy plasmids and tet(M) on conjugative transposons (Tn916–Tn1545 family) contributes to the spread of these determinants (Chopra and Robert 2001). One isolate carried both tet(K) and tet(M) genes. The carriage of multiple tet genes was commonly found in individual staphylococci species (Domínguez et al. 2002). The mecA gene, encoding resistance to methicillin, was detected in 45 out of 49 (91.8%) clinical bacteremic CoNS isolates collected in our center during the studied period. Indeed, mecA is carried by small-size SCCmec types, such as SCCmec IV, which is present in isolates of diverse genetic backgrounds and is presumed to be mobile in the
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environment. This mobility may be partially responsible for the spread of mecA and the rise in nosocomial methicillinresistant staphylococcal infections (Noto and Archer 2006). A close correspondence between resistance and PCR results was found for erythromycin, gentamicin, and tetracycline. Instead, this correlation was not observed for two isolates that carry mecA gene in the presence of oxacillin as previously described by Martineau et al. (2000) that reported the occurrence of S. aureus and S. epidermidis strains with the mecA gene but susceptible to oxacillin. The heterogeneous nature of methicillin resistance suggests that numerous factors could explain the sensitive phenotype in these strains, such as the regulation of the expression of mecA (Martineau et al. 2000). However, this could be associated also to an extreme heterogeneous expression of resistance combined in some cases to oxacillin SCCmec excision (Forbes et al. 2008). The most prevalent SCCmec types were IV (31% of strains) and III (24.5% of strains), identified only among S. epidermidis isolates. Type IV is also predominant in an earlier study (Wisplinghoff et al. 2003), but type III is the most prevalent in other studies (Chung et al. 2004; Machado et al. 2007). SCCmec type IV was the SCCmec more frequently acquired by S. epidermidis, which is in accordance with the enhanced mobility of this type of SCCmec already observed in S. aureus (Robinson and Enright 2003). S. epidermidis may be better adapted, due to an earlier contact with SCCmec, or may have the capacity to adapt faster to this piece of foreign DNA than S. aureus (Miragaia et al. 2007). Seventeen isolates (38%) were not typeable. Most of them were identified as S. haemolyticus in our study and as S. hominis in the study performed by Machado et al. (2007). The relatively large number of nontypeable SCCmec among our collection may be indicative of a higher diversity among the SCCmec carried by MRCoNS than among the SCCmec carried by MRSA. It may be due to the acquisition of novel SCCmec structures through rearrangement and recombination events (Zhang et al. 2005; Chung et al. 2004). A higher diversity of SCCmec cassettes harbored by MRCoNS compared to that of the cassettes harbored by S. aureus has been reported (Jamaluddin et al. 2008). In this study, isolates with SCCmec type III or IV were found to be resistant to nonβ-lactam antibiotics and harboring combination of the resistance genes tested. According to the literature, isolates containing SCCmec III contain a large number of resistance genes, but most SCCmec type IV in S. aureus and in CoNS strains remain susceptible to non-β-lactam antibiotics (Ito et al. 1999, 2001). It is noteworthy that SCCmec types in S. epidermidis appear to have occurred over the years. The majority of isolates of SCCmec type III were collected in 2006 and 2007, and the majority of isolates of SCCmec type IV were collected in 2005 and 2006.
In our study, a wide diversity of isolates was observed by PFGE and substitution of S. epidermidis clones appears to have occurred over the years (Table 1). The molecular characterization of CoNS isolates by PFGE and SCCmec typing reveals a high degree of genetic diversity, especially in S. epidermidis (Hanssen and Sollid 2007; Van Pelet et al. 2003). Nosocomial dissemination of S. epidermidis strains have been demonstrated by many authors and in many clinical wards (Klingenberg et al. 2004). This genetic diversity may be caused by the need for isolates to adapt to different environments in hospital setting leading to increased frequency of horizontal gene transfer and dissemination of mobile genetic elements. We believe that a reservoir of antimicrobial genes and SCCmec variants is being produced in S. epidermidis and subsequently transferred to S. aureus and to other staphylococcal species. In conclusion, our results confirmed the non-typeability of all studied S. haemolyticus and S. hominis methicillinresistant strains, the high prevalence of SCCmec IV and III in S. epidermidis strains harboring combinations of the resistance genes tested and the high genetic diversity among MRCoNS strains. Acknowledgment We would like to thank T. Ito (Japan) and R. Leclercq (France) for kindly providing control strains, included in this study and H. de Lencastre (Portugal) for providing the BioNumerics software.
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