|Year : 2016 | Volume
| Issue : 3 | Page : 187-191
Phenotypic characterization of macrolide and lincosamide resistance patterns in clinical isolates of staphylococci
Ribekha Zachariah, Sreekanth Basireddy, Vasanti Kabra, Manisha Singh, Salmaan Ali, Ahmed Sardar
Department of Microbiology, SVS Medical College, Yenugonda, Mahabubnagar, Telangana, India
|Date of Web Publication||10-Oct-2016|
Department of Microbiology, SVS Medical College, Yenugonda, Mahabubnagar - 509 001, Telangana
Source of Support: None, Conflict of Interest: None
Introduction: The macrolide, lincosamides, and streptogramin (MLS) group of antibiotics are one of the few alternative antibiotics available for the treatment of methicillin-resistant staphylococcal isolates. Clinical failures with these antibiotics have been reported because of the usage of these drugs in improperly characterized and tested isolates. The purpose of this study was to know the prevalence of different phenotypes in our area, with an emphasis on inducible resistance detection.
Materials and Methods: A total of 100 methicillin-resistant staphylococcal isolates were included in the study. Phenotypic characterization was done by using erythromycin and clindamycin disks kept at a distance of 15 mm on the Muller Hinton agar plate and incubating at 24 h.
Results: Among 100 isolates, 23 were sensitive to both erythromycin and clindamycin (S phenotype). Thirty-one isolates were resistant to erythromycin but sensitive to clindamycin (MS phenotype). Inducible resistance (iMLSB) was seen in 26 of the total isolates. Isolates which were resistant to both the antibiotics numbered 19 (cMLSB). There was only one isolate which was sensitive to erythromycin but resistant to clindamycin (L phenotype).
Conclusion: MLS group of antibiotics is still an effective group of antibiotics against methicillin-resistant staphylococcal isolates, and routine testing for the inducible resistance in these isolates will help in the prevention of therapeutic failure and for the better management of the patient.
Keywords: Inducible clindamycin resistance, MLSB, methicillin-resistant coagulase-negative staphylococci, methicillin-resistant Staphylococcus aureus, MS phenotype
|How to cite this article:|
Zachariah R, Basireddy S, Kabra V, Singh M, Ali S, Sardar A. Phenotypic characterization of macrolide and lincosamide resistance patterns in clinical isolates of staphylococci. J NTR Univ Health Sci 2016;5:187-91
|How to cite this URL:|
Zachariah R, Basireddy S, Kabra V, Singh M, Ali S, Sardar A. Phenotypic characterization of macrolide and lincosamide resistance patterns in clinical isolates of staphylococci. J NTR Univ Health Sci [serial online] 2016 [cited 2019 Dec 6];5:187-91. Available from: http://www.jdrntruhs.org/text.asp?2016/5/3/187/191847
| Introduction|| |
Macrolides, lincosamides, and streptogramin (MLS) antibiotics are chemically distinct group of antibiotics with a similar mode of action. All these antibiotics act on 50S ribosomal subunit and, thus, inhibit the bacterial protein synthesis. Though streptogramins are not widely used, the remaining two groups are used extensively in the treatment of gram-positive bacterial infections, especially in penicillin allergic patients. Widespread use of these antibiotics has led to an increased resistance in these groups of antibiotics.,, Bacteria show resistance to these antibiotics predominantly by target site modification through methylation or by efflux mechanism, the third mechanism being drug inactivation which is considered to be rare.
Target site modification is mediated by the enzymatic demethylation of the adenine residue in the 23S rRNA component of 50S ribosomal subunit. The erm gene which codes for methylase enzyme is responsible for this methylation. Because of the overlapping binding sites of the macrolides, lincosamides, and streptogramin B, this methylation confers cross resistance to all these groups of antibiotics showing MLSB phenotype. In staphylococci, erm(A) and erm(C) genes are typically involved whereas other erm genes play a role in different bacteria. This plasmid-borne erm gene can be expressed constitutively (constitutive MLSB phenotype) or inducibly (iMLSB phenotype). In constitutive expression, active methylase is produced even in the absence of an inducer antibiotic like erythromycin, whereas in inducible expression, this happens only in the presence of an inducer.
The second mechanism is antibiotic efflux which is mediated by ABC transporters encoded by msr(A) gene. Here we typically observe resistance to macrolides and streptogramins, with no effect on lincosamides (MS phenotype). The third mechanism of drug modification is mediated by lnu(A) or lnu(B) gene which code for lincosamide nucleotidyltransferases, with resistance to lincosamides only but not to the other groups.
Constitutive MLSB phenotypes can be easily detected during routine laboratory testing where the organism readily shows resistance to both erythromycin and clindamycin. But the inducible phenotype is often missed unless the inducer antibiotic (erythromycin) is placed adjacent to clindamycin. In inducible resistance, when erythromycin is placed adjacent to clindamycin, the D zone formation around clindamycin disk (blunting or flattening of clindamycin sensitivity zone toward erythromycin) is observed. If both the disks are placed far apart, clindamycin shows clear zone of sensitivity with only erythromycin showing resistance. Detecting this iMLSB phenotype is very important in a clinical setup as there is a chance of development of constitutive mutants which leads to the treatment failure if clindamycin is used in these patients. So, in our study, we have concentrated mainly on detecting this phenotype.
| Materials and Methods|| |
A total of 100 non-duplicate, randomly chosen methicillin-resistant staphylococcal isolates obtained from various clinical specimens were included in the study [50 methicillin-resistant Staphylococcus aureus (MRSA) and 50 methicillin-resistant coagulase-negative staphylococci (MRCoNS)]. Methicillin resistance was detected by using the cefoxitin disk diffusion testing (30 µg). In macrolide and lincosamides discordant strains, detection of the inducible resistance (D test) was done as per CLSI guidelines by keeping the erythromycin (15 µg) and clindamycin (2 µg) at a distance of 15 mm from edge to edge and incubating at 37°C for 24 h. Flattening of the zone of inhibition in between the two disks indicates clindamycin resistance.
Different phenotypes observed were appreciated and interpreted as follows:
- Sensitive (S) phenotype — both erythromycin and clindamycin sensitive.
- MS phenotype — erythromycin resistant and clindamycin sensitive.
- iMLSB phenotype — erythromycin resistant with D zone around clindamycin.
- cMLSB phenotype — both erythromycin and clindamycin resistant.
- L phenotype — erythromycin sensitive but lincosamide resistant.
| Results|| |
Overall, among 100 isolates, 23 (23%) were sensitive to both erythromycin and clindamycin. Thirty-one isolates (31%) showed resistance to erythromycin but were sensitive to clindamycin indicating MS phenotype [Figure 1]. Inducible resistance (iMLSB) was seen in 26 of the total isolates (26%) [Figure 2]. Isolates which were resistant to both the antibiotics accounted for 19% (cMLSB) [Figure 3]. There was only one isolate which was sensitive to erythromycin but resistant to clindamycin (L phenotype) [Figure 4].
When these phenotypes were subgrouped to species level, sensitive (S) phenotype was observed in MRSA and MRCoNS in 22% and 24% isolates, respectively. S. aureus showed predominant inducible resistance (34%) when compared to coagulase-negative staphylococci (18%). Constitutive resistance was higher in MRCoNS (22%) when compared to MRSA (16%). MS phenotype was also observed more commonly in MRCoNS (34%) than in MRSA (28%) [Table 1] and [Figure 5].
| Discussion|| |
Emergence of methicillin resistance in staphylococcal isolates is a serious threat to public health with only few therapeutic alternatives leftover to treat them. The MLSB group of antibiotics is one such alternative for these infections. Among them, clindamycin is considered the drug of choice because of excellent pharmacokinetic properties, good oral bioavailability, low cost, good tissue penetration, accumulation in abscesses, and its tolerability. Clindamycin also suppress the production of Panton-Valentine leukocidin toxin and toxic-shock syndrome toxin 1 (TSST 1) by staphylococci, which is why it is chosen over the other drugs in the treatment of life-threatening staphylococcal infections.
But extensive use of this drug has also created selective pressure with organisms becoming increasingly resistant to this antibiotic. Also, organisms with inducible resistance are increasingly being isolated. Treatment of these inducible resistant organisms with a non-inducible drug like clindamycin can lead to selection of constitutive mutants at frequencies of 107 colony-forming units (CFU). Because of these mutants, there is a chance for treatment failure, especially in serious infections where the bacterial load is very high. Case reports of treatment failures have been reported in such patients. That is why CLSI recommends that laboratories should report D test positive isolates as resistant to clindamycin.
Though clindamycin is used in the treatment of methicillin-sensitive staphylococci too, in our study, we have concentrated only on methicillin-resistant isolates as there are many alternatives available in the treatment of methicillin-sensitive strains.
In the present study, 22% of the MRSA were sensitive to both erythromycin and clindamycin. This finding is similar to the study conducted by Angel et al.,  where 24% of the MRSA were sensitive to both. In Kumar et al.'s  study, 28% of the MRCoNS were sensitive to both erythromycin and clindamycin, which is comparable to our study where 24% of the MRCoNS were sensitive. In our study, MS phenotype was observed in 28% of the MRSA, which is similar to the report of Deotale et al., where MS phenotype accounted for 24.3% of the MRSA. The present study showed 34% of MRCoNS with MS phenotype, which is slightly higher than that of MRSA. There is a very high variability in the rate of occurrence of MS phenotypes in different studies ranging from 0 to 72%., Together, clindamycin showed sensitivity in 50% of the total MRSA isolates and 58% of the total MRCoNS isolates (both S and MS phenotypes are considered sensitive) retaining its potential against such isolates.
In our study, cMLSB phenotype with resistance to both the antibiotics was observed in 16% and 22% of MRSA and MRCoNS isolates, respectively, which is similar to the study conducted by Samanth et al.,  where MRSA and MRCoNS showed cMLSB phenotype in 23% and 22%, respectively.
The very important phenotype, which is the core phenotype of this study, is inducible MLSB phenotype which accounted for 34% of total MRSA. This is in accordance with many other studies including Deotale et al.  (27.6%), Mohanasoundaram et al. (28%), Gadepalli et al. (30%), Braun et al. (30%), and Samanth et al.  (36%). This inducible phenotype was relatively less commonly seen in MRCoNS (only 18%) when compared with MRSA (34%). Our findings are similar to those of Kumar et al.  who found that only 18.8% of MRCoNS were D test+ whereas 75% of MRSA accounted for the same.
The least commonly seen phenotype was L phenotype, where the isolates are sensitive to erythromycin but resistant to lincomycin (another lincosamide), which is mediated by lnu gene. But in vitro, susceptibility to clindamycin appears sensitive though the antibacterial activity is diminished. But this L phenotype with clindamycin resistance and erythromycin sensitive is also seen as a part of cfr gene mediated multidrug resistance phenotype which is considered to be very rare.
Statistical analysis was done using Chi-square test for goodness of fit. Using this test it was found that MRSA and MRCoNS were independent of each other at 5% level of significance. Biologically, iMLSB in MRSA and MRCoNS was significantly different.
Though the incidence of these different phenotypes varies from place to place, knowledge of the occurrence of these different phenotypes is very important in a clinical setup in prescribing the appropriate treatment to the patients. Detection of inducible resistance should be practiced routinely in a clinical laboratory so as to avoid false-sensitive results and the D test+ isolates should be reported as resistance especially in severe and life-threatening infections.
| Conclusion|| |
To conclude, knowledge of circulating phenotypes of MLSB group of antibiotics is very important in starting the empirical therapy. Inducible resistance to clindamycin should be detected routinely in the microbiology laboratories by using the simple D test methodology. By using proper diagnostic methodologies and the appropriate antibiotic for the treatment of these methicillin-resistant isolates will help in not only decreasing the mortality and morbidity of patients but also in preserving the integrity of the few leftover alternatives for future use.
| References|| |
Drinkovie D, Fuller ER, Shore KP, Holland DJ, Ellis-Pegler R, Clindamycin treatment of Staphylococcus aureus
expressing inducible clindamycin resistance. J Antimicrob Chemother 2001;48:315-6.
Siberry GK, Tekle T, Carroll K, Dick J. Failure of clindamycin treatment of methicillin-resistant Staphylococcus aureus
expressing inducible clindamycin resistance in vitro
. Clin Infect Dis 2003;37:1257-60.
Rao GG. Should clindamycin be used in treatment of patients with infections caused by erythromycin-resistant staphylococci? J Antimicrob Chemother 2000;45:715.
Leclercq R. Mechanisms of resistance to macrolides and lincosamides nature of the resistance elements and their clinical implications. Clin Infect Dis 2002;34:482-92.
Kasten MJ. Clindamycin, metronidazole, and chloramphenicol. Mayo Clin Proc 1999;74:825-33.
Stevens DL, Ma Y, Salmi DB, McIndoo E, Wallace RJ, Bryant AE. Impact of antibiotics on expression of virulence-associated exotoxin genes. J Infect Dis 2007;195:202-11.
Angel MR, Balaji V, Prakash JA, Brahmandathan KN, Mathews MS. Prevalence of inducible clindamycin resistance in gram positive organisms in a tertiary care centre. Indian J Med Micobiol 2008; 26:262-4.
Kumar S, Umadevi S, Joseph N, Kali A, Easow J, Srirangaraj S, et al.
Detection of inducible clindamycin resistance in Staphylococcus aureus
and coagulase negative staphylococci- a study from South India. Internet J Microbiol 2010;9:2.
Deotale V, Mendiratta DK, Raut U, Narang P. Inducible clindamycin resistance in Staphylococcus aureus
isolated from clinical samples. Indian J Med Microbiol 2010;28:124-6.
Jenssen WD, Thakkervaria S, Dubin DT, Weinstein MP. Prevalence of macrolides-lincosamides-streptogramin-B resistance and erm
gene classes among clinical strains of Staphylococci and Streptococci. Antimicrob Agents Chemother 1987;31:883-8.
Tiwari S, Sahu M. Prevalence of inducible Clindamycin resistance in Staphylococcal isolates in a tertiary care hospital in Odisha. IJRRMS 2012;25-8.
Samant SA, Pai CG. Inducible Clindamycin Resistance among Clinical Isolates of Staphylococci. IJRPBS 2013;4:522-5.
Mohanasoundaram KM. The prevalence of inducible clindamycin resistance among gram positive cocci from various clinical specimens. JCDR 2011;5:38-40.
Gadepalli R, Dhawan B, Mohanty S, Kapil A, Das BK, Chaudhry R. Inducible clindamycin resistance in clinical isolates of Staphylococcus aureus
. Indian J Med Res 2006;123:571-3.
Braun L, Craft D, Williams R, Tuamokumo F, Ottolini M. Increasing clindamycin resistance among methicillin resistant Staphylococcus aureus
in 57 northeast United States military treatment facilities. Pediatr Infect Dis J 2005;24:622-6.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]