|Year : 2014 | Volume
| Issue : 1 | Page : 49-52
Prevalence of inducible clindamycin resistance among community-associated staphylococcal isolates in central Serbia
AD Aleksandra1, MS Misic2, ZV Mira3, NM Violeta3, IT Dragana3, BM Zoran4, VS Dejan5, SD Milanko5, BD Dejan6
1 Department of Microbiology, Institute of Medical Microbiology and Immunology, University of Pristina, Kosovska Mitrovica, Serbia
2 Public Health Institute Vranje, Serbia
3 Public Health Institute, Kragujevac, Serbia
4 Institute of Medical Statistics and Informatics, University of Belgrade, Serbia
5 Veterinary Specialized Institute Kraljevo, Kraljevo, Serbia
6 Public Health Institute, Kragujevac; Center for Molecular Medicine and Stem Cell Research, University of Kragujevac, Kragujevac, Serbia
|Date of Submission||06-Feb-2013|
|Date of Acceptance||16-Sep-2013|
|Date of Web Publication||4-Jan-2014|
B D Dejan
Public Health Institute, Kragujevac; Center for Molecular Medicine and Stem Cell Research, University of Kragujevac, Kragujevac
Source of Support: This research was supported by the Serbian
Ministry of Education and Science, project III 41010-2,, Conflict of Interest: None
The emergence of resistance to most antimicrobial agents in staphylococci indicates the need for new effective agents in the treatment of staphylococcal infections. Clindamycin is considered to be one safe, effective and less costly agent. We analysed 482 staphylococcal isolates. Detection of inducible clindamycin resistance was performed by the D-test, while the presence of methylases genes: erm (A), erm (B) and erm (C), as well as, macrolide efflux gene mef was determined by polymerase chain reaction. Inducible clindamycin resistance phenotype was significantly higher in Staphylococcus aureus (S. aureus) strains then in coagulase-negative staphylococci (CNS). Among analysed S. aureus isolates, the predominance of the erm (C) gene, followed by the erm (A) gene were detected. These results indicate that the D-test should be routinely performed on each staphylococcal isolates.
Keywords: Coagulase-negative staphylococci, inducible clindamycin resistance, Staphylococcus aureus
|How to cite this article:|
Aleksandra A D, Misic M S, Mira Z V, Violeta N M, Dragana I T, Zoran B M, Dejan V S, Milanko S D, Dejan B D. Prevalence of inducible clindamycin resistance among community-associated staphylococcal isolates in central Serbia. Indian J Med Microbiol 2014;32:49-52
|How to cite this URL:|
Aleksandra A D, Misic M S, Mira Z V, Violeta N M, Dragana I T, Zoran B M, Dejan V S, Milanko S D, Dejan B D. Prevalence of inducible clindamycin resistance among community-associated staphylococcal isolates in central Serbia. Indian J Med Microbiol [serial online] 2014 [cited 2019 Aug 23];32:49-52. Available from: http://www.ijmm.org/text.asp?2014/32/1/49/124304
| ~ Introduction|| |
The increasing prevalence of resistance to most antimicrobial agents in staphylococci, especially spread of resistant strains in the community, signify the need for new effective agents to treat staphylococcal infections. Widespread use of macrolide-lincosamide-streptogramin (MLS) antibiotics, has led to an increase in a number of staphylococci acquiring cross-resistance to MLS antibiotics.  This cross-resistance to MLS antibiotic (MLS resistance) in staphylococci, is generally attributable to one of two mechanisms.
First is an active efflux, due to energy-dependent pump, which expels antimicrobial agents from the bacterial cell. Efflux mechanism is encoded by mrs (A) gene and confers resistance to macrolides and type B streptogramin, but clindamycin remains active (MS B resistance). Second mechanism is modification of the drug-binding site on the bacterial ribosome, mediated by ribosomal methylases, which leads to the reduced binding of MLS antibiotics. Ribosomal methylases are encoded by erm genes (erm (A) or erm (C) in staphylococci) and results in resistance to macrolides, lincosamides and type B streptogramin (MLS B resistance). ,
Phenotypic expression of this resistance can be constitutive (cMLS B resistance phenotype) and inducible (iMLS B resistance phenotype). In inducible expression, the bacteria produce inactive methylase mRNA that is unable to translate in to ribosomal methylase, but becomes active only in the presence of a strong inducer, such as erythromycin. By contrast, in constitutive expression, active methylase mRNA is produced in continuity, and in the absence of an inducer. In vitro, staphylococcal isolates with cMLS B phenotypes are resistant to all MLS B antibiotics, whereas those with iMLS B phenotypes demonstrate resistance to macrolides, while appearing susceptible to lincosamides and type B streptogramin. ,
To detect iMLS B resistant strains, there are special disk-approximation test that involve the placement of an erythromycin disk in close proximity to a disk containing clindamycin. As the erythromycin diffuse through the agar, resistance to the clindamycin is induced, giving a D shape to the zone of clindamycin disk. 
Published data of inducible clindamycin resistance among pathogen staphylococcal isolates in Serbia, to the best of our knowledge, are missing. Because of that, the present study was aimed, to investigate MS B and MLS B resistance and to record the current trend in regard to the prevalence and distribution of inducible clindamycin resistance among community-associated (CA) Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CNS) in region of central Serbia.
| ~ Materials and Methods|| |
We analysed 482 staphylococcal isolates, collected from the Public Health Institute, Kragujevac. The Public Health Institute in Kragujevac provides microbiology laboratory services to primary health care centre (e.g. general practitioner, preventative care) in an outpatient setting. Staphylococcal isolates were labelled as community-associated staphylococcal isolates if the source patient had none of the following risk factors: a history of hospitalisation or surgery within one year prior to the date of specimen collection, haemodialysis and residence in long-term care facility. Staphylococcal isolates were collected from different patient samples (pus, wound swab, aspirates, eye swab, sputum, nasal and throat swab). Strains were identified using conventional bacteriological methods, and their susceptibility testing was first performed by the standard disk diffusion method on Mueller-Hinton agar (MHA), according to the standards of Clinical and Laboratory Standards Institute (CLSI).  Each erythromycin-resistant clindamycin susceptible isolates were further tested for inducible resistance with D-test, as described in the CLSI recommendations. Isolates with the MS B resistance, demonstrated circular clindamycin zone, while isolates with the iMLS B phenotype, demonstrated a flattening of the clindamycin zone. Isolates resistant to both erythromycin and clindamycin confers to cMLS B resistance phenotype.
Detection of resistance genes
Polymerase chain reaction (PCR) was performed on 20 S. aureus strains displaying MLS B resistance phenotype. Genomic DNA was extracted from staphylococcal overnight cultures using a QIAamp DNA Mini Kit (Qiagen) following protocol for isolation of genomic DNA from Gram-positive bacteria. Presence of erythromycin resistance methylase erm genes erm (A), erm (B) and erm (C), as well as macrolide efflux gene mef was determined by PCR using the primer pairs as described by Lim et al.,  All primers were synthesised by Invitrogen. Each reaction was carried out in a final volume of 50 μl using 2 μl of template, 1 μl of each primer (100 μM), 25 μl of Maxima® Hot Start Green PCR Master Mix (Fermentas) and 21 μl of PCR grade water. PCR conditions consisted of a denaturation step (4 min at 94°C), followed by 35 cycles at 94°C for 30 s of denaturation, 30 s of annealing at 52°C, 1 min of elongation at 72°C and a final extension step (5 min at 72°C). Amplified products were detected by gel electrophoresis on E-Gel iBase (Invitrogen) in 2% (w/v) agarose gels (E-Gel® 2%, Invitrogen) and visualised in Gel Doc XR system (Bio-Rad). The size of the PCR products was determined by comparison with a 100-1000 bp molecular weight marker O'GeneRuler™ 100 bp DNA Ladder (Fermentas). Positive control strains used in this study were: Escherichia coli NM522 (ermA), Bacillus subtilis 168 (ermB), Bacillus subtilis BD170 (ermC), Streptococcus pyogenes 02C1064 (mef).
Data were analysed using Chi-squared test (χ2) and the probability level of 0.05 was considered to be statistically significant.
| ~ Results|| |
Our study included 482 staphylococcal isolates, of which 395 (82%) were S. aureus and 87 (18%) were CNS. Of the 482 staphylococcal isolates, 191 (40%) were resistant to erythromycin (ERY-R).
In order to accurate insight into the possibility of misinterpretation of staphylococcal resistance to clindamycin, we performed D-test and found that majority of ERY-R S. aureus isolates, display iMLS B phenotypes, while most of ERY-R CNS, show MS B phenotypes [Table 1]. Truly susceptible isolates (MS B phenotype) were significantly higher in CNS strains then in S. aureus0 strains (χ2 = 13.19; P < 0.001). Falsely susceptible isolates (iMLS B phenotype) were significantly higher in S. aureus strains then in CNS strains (χ2 = 16.52; P < 0.001). However, no statistically significant difference of truly resistant isolates (cMLS B phenotype) was observed between S. aureus and CNS isolates (χ2 = 0.20; P = 0.655).
|Table 1: Distribution of the MSB, iMLSB and cMLSB resistance phenotypes among erythromycin resistant S. aureus and CNS isolates|
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The predominant genes associated with macrolide resistance among S. aureus were the erm (C) and the erm (A), which were detected in 55% (11/20) and 40% (8/20) of the isolates, respectively [Table 2]. The erm (C) determinant was found in six (60%) cMLS B S. aureus isolates and one of these strains also contained erm (A). Similarly, half of iMLS B strains harboured erm (C) gene. The distribution of erm (A) gene in cMLS B and iMLS B S. aureus isolates was identical since both phenotypes carried this gene in 40% of the cases. Two of the MLS B isolates did not harbour any of the resistance genes. Among tested isolates erm (B) and mef genes were not detected [Figure 1].
|Figure 1: PCR of S. aureus isolates. Lines 1 and 2: Amplifi ed product (572 bp) of erm (C) gene. Line 3: Amplifi ed product (421 bp) of erm (A) gene. Line 4: Molecular weight marker|
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|Table 2: Distribution of the erm and mef genes among 20 MLSB S. aureus isolates|
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| ~ Discussion|| |
One of the major concerns with regard to the therapeutic use of clindamycin in staphylococcal infection is the possible presence of inducible resistance to clindamycin, and subsequent clinical failure of therapy. 
Simple laboratory testing, (i.e. erythromycin-clindamycin 'D-zone' test) can separate strains that have the genetic potential to become resistant during therapy from strains that are truly susceptible to clindamycin. Without the double-disk test, all staphylococcal isolates with iMLS B would have been mistakenly interpreted as clindamycin-susceptible. On the other side, to categorically consider all erythromycin resistant staphylococci as clindamycin resistant would deny potentially safe and effective therapy for patients infected with isolates that carry only the export mechanism. 
For instance, in our study we had 24% truthfully (MS B ) and 50% untruly (iMLS B ) clindamycin susceptible S. aureus isolates, resulting in an underestimated clindamycin resistance rate of 26% (cMLS B ) instead of 76% (iMLS B and cMLS B ) among S. aureus and similarly, 30% instead 48% among CNS [Table 1].
Also, our results support the fact that erm (C) and erm (A) genes predominated within erythromycin-resistant S. aureus isolates with MLS B phenotype. , Yet, whereas, some of previous reports demonstrated prevalence of erm (A) genes,  other, similar to our study, showed that the erm (C) gene was more common than erm (A) in MLS B S. aureus strains. 
For these reasons it is necessary to examine the prevalence of iMLS B , especially in a setting where methicillin resistance leads physicians to prescribed different macrolides or lincosamides empirically.  This prevalence may change over time with the emergence of strains with different sensitivity patterns, so periodic surveys should be performed if testing is not routine.
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[Table 1], [Table 2]