Users Online: 753
Home Print this page Email this page
Home About us Editorial board Search Browse articles Submit article Ahead of Print Instructions Subscribe Contacts Login 


 
Previous article Browse articles Next article 
ORIGINAL ARTICLE
Adv Biomed Res 2016,  5:62

Distribution of erm genes among Staphylococcus aureus isolates with inducible resistance to clindamycin in Isfahan, Iran


1 Department of Microbiology, Faculty of Bioscience, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
2 Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Endodontics, School of Dentistry, Zahedan University of Medical Sciences, Zahedan, Iran
4 Cellular and Molecular Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran
5 Department of Medical Bacteriology and Virology, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
6 Department of Microbiology, School of Medicine, Ahvaz University of Medical Sciences, Ahvaz, Iran

Date of Submission30-Nov-2014
Date of Acceptance24-May-2015
Date of Web Publication22-Mar-2016

Correspondence Address:
Dr. Seyed Asghar Havaei
Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2277-9175.179184

Rights and Permissions
  Abstract 

Background: The rising frequency of methicillin resistant Staphylococcus aureus (MRSA) has led to an increased use of antibiotics such as macrolide, lincosamide, streptogramin B (MLSB) for the treatment of S. aureus infections. Resistance to MLSBin S. aureus is commonly encoded by erm genes, which can be constitutive MLSB (cMLSB) or inducible MLSB (iMLSB). The purpose of this study was to determine the frequency of cMLSB, iMLSB, and MS phenotypes using D-test and polymerase chain reaction (PCR) methods.
Materials and Methods: A total of 215 isolates of S. aureus were collected from January 2010 to May 2012 from Al-Zahra Hospital in Isfahan. PCR was performed for detection of mecA gene on all isolates using specific primers. The frequency of MLSB-resistant isolates was determined using D-test, and then a multiplex PCR was performed for detection of ermA, ermB, and ermC genes.
Results: Among 215 S. aureus isolates examined, 82 (40.9%) were MRSA, and iMLSB, cMLSB, and MS resistance phenotypes had a frequency of 9 (4.18%), 58 (26.9%), and 11 (5.1%), respectively. Among nine isolates with iMLSBresistance phenotype, four isolates contained ermC gene, two isolates ermB gene, and one isolate ermA gene. Two isolates did not have any erm gene.
Conclusion: In the current study, cMLSBwas the most frequent phenotype and ermC was the most common gene in iMLSBresistant phenotypes.

Keywords: Clindamycin, D-test, erm genes, inducible resistance, Staphylococcus aureus


How to cite this article:
Ghanbari F, Ghajavand H, Havaei R, Jami MS, Khademi F, Heydari L, Shahin M, Havaei SA. Distribution of erm genes among Staphylococcus aureus isolates with inducible resistance to clindamycin in Isfahan, Iran. Adv Biomed Res 2016;5:62

How to cite this URL:
Ghanbari F, Ghajavand H, Havaei R, Jami MS, Khademi F, Heydari L, Shahin M, Havaei SA. Distribution of erm genes among Staphylococcus aureus isolates with inducible resistance to clindamycin in Isfahan, Iran. Adv Biomed Res [serial online] 2016 [cited 2019 Mar 23];5:62. Available from: http://www.advbiores.net/text.asp?2016/5/1/62/179184


  Introduction Top


Staphylococcus aureus is one of the most frequent pathogens that cause both community and hospital-acquired infections worldwide. Development of drug resistance in S. aureus has led to the use of older antibiotics such as macrolide, lincosamide, and streptogramin B (MLSB) antibiotic.[1],[2] However, extensive use of these antibiotics in serious staphylococcal infections has caused the emergence of S. aureus resistant to MLSB antibiotics.[3] There are three different mechanisms of resistance to MLSB antibiotics including: (1) Active efflux mechanism encoded by msr gene, (2) drug inactivation encoded by lun gene and (3) ribosomal binding site modification (by methylation or mutation in the 23s rRNA gene) encoded by erm genes (ermA, ermB, ermC, and ermF) among which, ermA and ermC are predominant genes responsible for resistance to MLSB antibiotics in staphylococci, which can be constitutive or inducible.[4],[5],[6],[7],[8]In vitro, S. aureus isolates with constitutive MLSB (cMLSB) resistance are resistant to erythromycin and clindamycin but isolates with inducible MLSB (iMLSB) resistance are resistant to erythromycin and susceptible to clindamycin. In this condition, treatment of patients with clindamycin can lead to the emergence of resistant mutants to cMLSB from iMLSB-resistant strains and treatment failure.[3],[6] On the other hand, assigning all erythromycin-resistant S. aureus as clindamycin resistant strains may cause to avoid the use of clindamycin in the treatment of S. aureus infections. For this reason, careful screening of iMLSB-resistant strains is very important. While constitutive resistance is detectable by routine antimicrobial susceptibility tests, inducible resistance to clindamycin is not detectable by standard methods.[4],[5] For detection of iMLS-resistant strains, Clinical and Laboratory Standards Institute (CLSI) developed a phenotypic method called the double disk diffusion test (D-test).[9],[10],[11],[12] The aim of this study was to determine the frequency of inducible resistance to clindamycin using D-test and polymerase chain reaction (PCR) with specific primers to confirm the presence of the erm genes in these isolates.


  Materials and Methods Top


Bacterial strains and phenotypic testing

A total of 215 clinical isolates of S. aureus were collected from Al-Zahra Hospital in Isfahan from January 2010 to May 2012. Bacterial isolates were obtained from various clinical specimens including: Wound, blood, urine, sputum, etc., Early identification was performed based on Gram-staining and positive biochemical reactions such as catalase, coagulase, and DNase tests. D-test method was performed according to the CLSI guidelines using clindamycin (2 µg) and erythromycin (15 µg) disks (Himedia-India). For this purpose, suspensions of bacteria were prepared in the sterile saline (2 ml) equivalent to standard 0.5 McFarland and then two antibiotic disks placed on Muller-Hinton agar media in 15 mm distance (edge-to-edge). Plates were incubated at 35°C overnight. Strains with flat zone of growth inhibition of clindamycin near the erythromycin disk (D-shape) were classified as resistant phenotypes to iMLSB (D-test positive), while those with a circular zone were classified as MS resistant phenotypes (D-test negative) [Figure 1].
Figure 1: D-shape zone of growth inhibition around clindamycin disk (inducible macrolide, lincosamide, streptogramin B phenotype)

Click here to view


Molecular detection of mecA gene

DNA was extracted from 215 S. aureus isolates using Fermentas K0512 DNA kit (Fermentas-USA) in accordance with the manufacturer's protocol. PCR reaction was carried out for the amplification of the 310 bp fragment of mecA gene using primers as exhibited in [Table 1]. PCR amplification reaction mixture (25 μL) contained 4 µL of DNA template, 2.5 µL of PCR buffer (×10), 0.75 µL Mgcl2 (50 mM), 0.5 µL of dNTPs (10 mM), 1 µL of each primers (2 μL totally), 0.25 µL of Ex-Taq DNA polymerase (5u/µL) and 15 µL distill water. PCR conditions were as follows: Initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 30 s, and final extension at 72°C for 7 min.
Table 1: Primers used in this study

Click here to view


Multiplex polymerase chain reaction for erm gene

Multiplex PCR was performed for detection of erm gene in D-test positive isolates using specific primers for the ermA, B and C genes as exhibited in [Table 1]. Each PCR was performed in a final volume of 25 µL consisting of 5 µL of DNA template, 2.5 µL of PCR buffer (×10), 1 µL Mgcl2 (50 mM), 0.5 µL of dNTPs (10 mM), 0.75 µL of each primers (2 μL totally), 0.25 µL of Ex-Taq DNA polymerase (5 u/µL), 11.25 µL distill water. DNA was amplified on a thermocycler (Ependorf-Germany), and PCR conditions were as follows: Initial denaturation at 94°C for 10 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 60 s, followed by a final extension at 72°C for 10 min.


  Results Top


In this study, 215 isolates of S. aureus were collected from various clinical specimens, wound 53 (24.6%), blood 49 (22.79%), urinary tract infection 30 (13.9%), sputum 35 (16.27%), abscess 21 (9.76) and others 27 (12.55%), from Al-Zahra Hospital in Isfahan. The patient's average age was 47 years (ranged 1–88 years). The mecA gene screening in all isolates showed that 82 (40.9%) of the 215 tested isolates were methicillin resistant S. aureus (MRSA) and mecA positive [Figure 2]. Furthermore, double disk diffusion test results revealed that 134 (62.3%) of the isolates were susceptible to both clindamycin and erythromycin and 81 (37.7%) were shown to have four different resistance phenotypes in which 58 (26.9%) isolates were resistant phenotype to cMLSB(resistant to both erythromycin and clindamycin), 9 (4.18%) isolates were resistant phenotype to iMLSB (resistant to erythromycin and susceptible to clindamycin), 11 (5.1%) isolates were MS resistance phenotype (susceptible to clindamycin and resistant to erythromycin) and finally, 3 (1.39%) isolates were susceptible to erythromycin and resistant to clindamycin [Figure 3]. Among nine isolates with iMLSB resistance phenotype, 5 (55.5%) were MRSA. Nine staphylococcal isolates with iMLSB resistance phenotype were tested for the presence of the erm genes, the ermA gene in 1 (11.1%) isolate, the ermB gene in 2 (22.2%) isolates, the ermC gene in 4 (44.4%) isolates was detected and two isolates did not have any erm genes [Figure 3].
Figure 2: Gel electrophoresis of mecA gene. Lanes 1–5: 310 bp fragment, Lane 6: positive control of methicillin resistant Staphylococcus aureus Scientific Name Search  strains ATCC 33591, Lane 7: DNA Ladder100 bp

Click here to view
Figure 3: Gel electrophoresis of erm genes. Lane 1: ermA positive (421 bp), Lane 2 and 3: ermB positive (359 bp), Lane 4: ermC positive (572 bp), Lane 5: DNA Ladder100 bp

Click here to view



  Discussion Top


D-test results in our study demonstrated that 134 (62.3%) isolates were sensitive to both erythromycin and clindamycin; the frequency of cMLSB, iMLSB, and MS phenotypes were found to be 58 (26.9%), 9 (4.18%), and 11 (5.1%), respectively. In addition, the frequency of ermC, ermB, and ermA genes among isolates with iMLSB phenotype was determined to be 44.4%, 22.2%, and 11.1% respectively. Clindamycin due to its advantages including low-cost, low side effects, and good tissue penetration is used for the treatment of S. aureus infections. Although it is a good alternative in allergic patients instead of β-lactam antibiotics;[1],[9],[14],[15] however, excessive use of this antibiotic has an important role in bacterial resistance to clindamycin. Since the treatment of infected patients with resistant strains to iMLSB can lead to the expansion of constitutive resistance (cMLSB) and therapy failure with clindamycin, detection of resistant strains to iMLSB is important from other resistance phenotypes. Since the frequency of cMLSB, iMLSB, and MS phenotypes varies in different geographical areas, even among different hospitals, awareness of regional frequency of MLSB resistant isolates is important for laboratories to decide for performing the D-test routinely or reporting all erythromycin-resistant S. aureus as clindamycin resistant.[7],[10],[12],[16]

In the current study, 82 (40.9%) isolates were found to be MRSA that is, comparable with a study conducted by Seifi et al.[5] Also, 6.09% of MRSA isolates had resistant phenotype to iMLSB, which is lower than those reported by Shoja et al.[17] In the current study, 134 (62.3%) isolates were sensitive to both erythromycin and clindamycin and the frequency of cMLSB, iMLSB and MS phenotypes were found to be 58 (26.9%), 9 (4.18%), and 11 (5.1%) respectively. Similar results were reported by Aslanimehr et al.[18] In the present study, the frequency of cMLSB phenotype was higher than iMLSB phenotype. Similar results were obtained by Memarian et al.[19] and Mahesh et al.[20] In contrast, Reddy and Suresh found the frequency of iMLSB phenotype to be higher than cMLSB phenotype.[3] In our study, the frequency of MS resistance phenotype was shown to be higher than iMLSB phenotype, which was concordant to some previous studies.[3],[5],[7] Incidentally, we detected 3 (1.39%) isolates resistant to clindamycin and susceptible to erythromycin, similar results were also obtained by Coutinho et al.[10] In addition, Seifi et al. reported 6 (2.84%) S. aureus isolates with such a phenotype.[5] This phenotype can be created by lincosamide nucleotide transferase enzyme that only inactivates lincosamide (clindamycin). Therefore, we investigated erm gene distribution among isolates with iMLSB phenotype. Our results revealed the frequency of ermC, ermB, and ermA genes among isolates with iMLSB phenotype to be 44.4%, 22.2%, and 11.1%, respectively. Two isolates with iMLSB phenotype were negative in genotypic test.

It must be noted that the frequency of erm genes is variable in different studies. According to our findings, the ermC gene was the most prevalent gene, similar study was performed by Aktas et al. in Turkey,[7] while in a study conducted by Saderi et al. ermA gene was prevalent (60%) among erythromycin-resistant S. aureus.[2] An interesting point to notice in our study was the high frequency of ermB gene, Similar results were shown in some studies.[21],[22]


  Conclusion Top


This report has investigated the frequency of inducible resistance to clindamycin using D-test and PCR methods. This was the first study to investigate the frequency of MLSB phenotypes in Isfahan which demonstrated cMLSB resistance to be the most prevalent resistance phenotype, ermC gene as the most common gene among iMLS-resistant S. aureus and iMLSB phenotype having a low frequency. Therefore, we do not recommend the routine performance of D-test but since the frequency of different resistance phenotype may change through time with the emergence of strains with different antibiotic susceptibility patterns, it is recommended that local periodic survey be performed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Vivek JS, Rajesh GN, Mukesh S, Manpreet K, Manpreet K, Misra RN, et al. Prevalence of inducible clindamycin resistance among community- and hospital-associated Staphylococcus aureus isolates in a tertiary care hospital in India. Biomed Res 2011;22:465-9.  Back to cited text no. 1
    
2.
Saderi H, Emadi B, Owlia P. Phenotypic and genotypic study of macrolide, lincosamide and streptogramin B (MLSB) resistance in clinical isolates of Staphylococcus aureus in Tehran, Iran. Med Sci Monit 2011;17:BR48-53.  Back to cited text no. 2
    
3.
Reddy SP, Suresh R. Phenotypic detection of inducible clindamycin resistance among the clinical isolates of staphylococcus aureus by using the lower limit of inter disk space. J Microbiol Biotechnol Res 2012;2:258-64.  Back to cited text no. 3
    
4.
Zelazny AM, Ferraro MJ, Glennen A, Hindler JF, Mann LM, Munro S, et al. Selection of strains for quality assessment of the disk induction method for detection of inducible clindamycin resistance in staphylococci: A CLSI collaborative study. J Clin Microbiol 2005;43:2613-5.  Back to cited text no. 4
    
5.
Seifi N, Kahani N, Askari E, Mahdipour S, Naderi NM. Inducible clindamycin resistance in Staphylococcus aureus isolates recovered from Mashhad, Iran. Iran J Microbiol 2012;4:82-6.  Back to cited text no. 5
    
6.
Steward CD, Raney PM, Morrell AK, Williams PP, McDougal LK, Jevitt L, et al. Testing for induction of clindamycin resistance in erythromycin-resistant isolates of Staphylococcus aureus. J Clin Microbiol 2005;43:1716-21.  Back to cited text no. 6
    
7.
Aktas Z, Aridogan A, Kayacan CB, Aydin D. Resistance to macrolide, lincosamide and streptogramin antibiotics in staphylococci isolated in Istanbul, Turkey. J Microbiol 2007;45:286-90.  Back to cited text no. 7
    
8.
Schmitz FJ, Sadurski R, Kray A, Boos M, Geisel R, Köhrer K, et al. Prevalence of macrolide-resistance genes in Staphylococcus aureus and Enterococcus faecium isolates from 24 European university hospitals. J Antimicrob Chemother 2000;45:891-4.  Back to cited text no. 8
    
9.
Fiebelkorn KR, Crawford SA, McElmeel ML, Jorgensen JH. Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci. J Clin Microbiol 2003;41:4740-4.  Back to cited text no. 9
    
10.
Coutinho Vde L, Paiva RM, Reiter KC, de-Paris F, Barth AL, Machado AB. Distribution of erm genes and low prevalence of inducible resistance to clindamycin among staphylococci isolates. Braz J Infect Dis 2010;14:564-8.  Back to cited text no. 10
    
11.
Sedighi I, Yousefi Mashouf R, Pak N, Seif Rabiee MA. D-test method for detection of inducible clindamycin resistance in Staphylococcus aureus. Iran J Pediatr 2009;19:293-7.  Back to cited text no. 11
    
12.
O'Sullivan MV, Cai Y, Kong F, Zeng X, Gilbert GL. Influence of disk separation distance on accuracy of the disk approximation test for detection of inducible clindamycin resistance in Staphylococcus spp. J Clin Microbiol 2006;44:4072-6.  Back to cited text no. 12
    
13.
Lina G, Quaglia A, Reverdy ME, Leclercq R, Vandenesch F, Etienne J. Distribution of genes encoding resistance to macrolides, lincosamides, and streptogramins among staphylococci. Antimicrob Agents Chemother 1999;43:1062-6.  Back to cited text no. 13
    
14.
Lim HS, Lee H, Roh KH, Yum JH, Yong D, Lee K, et al. Prevalence of inducible clindamycin resistance in staphylococcal isolates at a Korean tertiary care hospital. Yonsei Med J 2006;47:480-4.  Back to cited text no. 14
    
15.
Schreckenberger PC, Ilendo E, Ristow KL. Incidence of constitutive and inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci in a community and a tertiary care hospital. J Clin Microbiol 2004;42:2777-9.  Back to cited text no. 15
    
16.
Lewis JS 2nd, Jorgensen JH. Inducible clindamycin resistance in Staphylococci: Should clinicians and microbiologists be concerned? Antimicrob Resist 2005;40:280-5.  Back to cited text no. 16
    
17.
Shoja S, Nahaei M, Nahaei M. Detection of inducible clindamycin resistance in Staphylococcus aureus and Staphylococcus epidermidis by using D-test. Pharma Sci 2009;15:1-8.  Back to cited text no. 17
    
18.
Aslanimehr M, Yaghobfar R, Peymani A. Detection of MLSB phenotypes and inducible clindamycin resistance in Staphylococcus aureus isolates in-patients of Qazvin and Tehran teaching hospitals. J Qazvin Univ Med Sci 2014;18:30-6.  Back to cited text no. 18
    
19.
Memariani M, Pourmand MR, Shirazi MH, Soltan Dallal MM, Abdolsamadi Z, Mardani N. The importance of inducible clindamycin resistance in enterotoxin positive S. aureus isolated from clinical samples. Tehran Univ Med J 2009;67:250-6. [Persian]  Back to cited text no. 19
    
20.
Mahesh CB, Ramakant BK, Jagadeesh VS. The prevalence of inducible and constitutive clindamycin resistance among the nasal isolates of staphylococci. J Clin Diagn Res 2013;7:1620-2.  Back to cited text no. 20
    
21.
Emaneini M, Eslampour MA, Sedaghat H, Aligholi M, Jabalameli F, Shahsavan S, et al. Characterization of phenotypic and genotypic inducible macrolide resistance in staphylococci in Tehran, Iran. J Chemother 2009;21:595-7.  Back to cited text no. 21
[PUBMED]    
22.
Zmantar T, Chaieb K, Ben Abdallah F, Ben Kahla-Nakbi A, Ben Hassen A, Mahdouani K, et al. Multiplex PCR detection of the antibiotic resistance genes in Staphylococcus aureus strains isolated from auricular infections. Folia Microbiol (Praha) 2008;53:357-62.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]


This article has been cited by
1 Understanding the impact of antibiotic therapies on the respiratory tract resistome: a novel pooled-template metagenomic sequencing strategy
Steven L. Taylor,Lex E. X. Leong,Fredrick M. Mobegi,Jocelyn M. Choo,Lucy D. Burr,Steve Wesselingh,Geraint B. Rogers
Multidisciplinary Respiratory Medicine. 2018; 13(S1)
[Pubmed] | [DOI]
2 Phenotypic and genotypic characterization of macrolide, lincosamide and streptogramin B resistance among clinical isolates of staphylococci in southwest of Iran
Reza Khashei,Yalda Malekzadegan,Hadi Sedigh Ebrahim-Saraie,Zahra Razavi
BMC Research Notes. 2018; 11(1)
[Pubmed] | [DOI]
3 Molecular Investigation of Methicillin-Resistant Staphylococcus aureus Strains Recovered from the Intensive Care Unit (ICU) Based on Toxin, Adhesion Genes and agr Locus Type Analysis
Sara Nasirian,Sara Saadatmand,Hossein Goudarzi,Mehdi Goudarzi,Hadi Azimi
Archives of Clinical Infectious Diseases. 2018; In Press(In Press)
[Pubmed] | [DOI]
4 Biofilm formation in erythromycin-resistant Staphylococcus aureus and the relationship with antimicrobial susceptibility and molecular characteristics
Xiang Sun,Zhi-wei Lin,Xiao-xiong Hu,Wei-ming Yao,Bing Bai,Hong-yan Wang,Duo-yun Li,Zhong Chen,Hang Cheng,Wei-guang Pan,Ming-gui Deng,Guang-jian Xu,Hao-peng Tu,Jun-wen Chen,Qi-wen Deng,Zhi-jian Yu,Jin-xin Zheng
Microbial Pathogenesis. 2018;
[Pubmed] | [DOI]
5 Molecular Characterization and Resistance Profile of Methicillin Resistant Staphylococcus aureus Strains Isolated from Hospitalized Patients in Intensive Care Unit, Tehran-Iran
Ramin Rashidi Nezhad,Seyed Mansour Meybodi,Razieh Rezaee,Mehdi Goudarzi,Maryam Fazeli
Jundishapur Journal of Microbiology. 2017; In press(In press)
[Pubmed] | [DOI]
6 Prevalence of Genotypes That Determine Resistance of Staphylococci to Macrolides and Lincosamides in Serbia
Milena Mišic,Jelena Cukic,Dejan Vidanovic,Milanko Šekler,Sanja Matic,Mihailo Vukašinovic,Dejan Baskic
Frontiers in Public Health. 2017; 5
[Pubmed] | [DOI]
7 Distribution of Genes Encoding Toxin, Adhesion, and Antibacterial Resistance Among Various SCCmec Types of Methicillin-Resistant Staphylococcus aureus Isolated From Intensive Care Unit, Tehran, Iran
Parnaz Abiri,Abbas Akhavan Sepahi,Hossein Goudarzi,Mehdi Goudarzi
Jundishapur Journal of Microbiology. 2017; In Press(In Press)
[Pubmed] | [DOI]



 

Top
Previous article  Next article
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed817    
    Printed16    
    Emailed0    
    PDF Downloaded205    
    Comments [Add]    
    Cited by others 7    

Recommend this journal