Detection of a Novel G2603T Mutation in cfr Harboring Linezolid-Resistant Staphylococcus haemolyticus: First Report from India

Introduction

Staphylococcus haemolyticus is an opportunistic bacterial pathogen that colonizes human skin and mucous membrane. It is the second most common coagulase-negative Staphylococci (CONS) isolated from clinical specimens and is associated with bloodstream infections related to intravascular catheters, skin and soft tissue infection, meningitis, endocarditis, and a variety of device-associated infections.^[1] It has an inherent ability to acquire and maintain exogenous genetic material or mobile genetic elements that encode for antimicrobial resistance.^[2] Hence, they are often multidrug resistant exhibiting resistance to antimicrobial classes such as beta lactams, macrolides, lincosamides, and streptogramins and more recently display reduced susceptibility to glycopeptides and oxazolidinones.^[3]

Linezolid is a synthetic bacteriostatic drug, belonging to oxazolidinone class of antibiotics and is active against various multidrug-resistant gram-positive pathogens, such as methicillin-resistant Staphylococci and vancomycin-resistant Enterococci. Linezolid inhibits protein synthesis by interacting with the 23S rRNA in the 50S ribosomal subunit.^[4,5] It is effective in the treatment of bacteremia, nosocomial pneumonia, and severe skin and soft tissue infections.^[6]

A year after its introduction, the first clinical linezolid-resistant Staphylococcus aureus strain appeared in 2001 and thereafter few reports were published from the United States and Europe.^[7] The first linezolid-resistant Staphylococcus haemolyticus (LRSH) was reported in 2009, since then a few strains have been reported from countries such as India, China, Brazil, Italy, and Spain.^[2] More recently due to its extensive use, linezolid resistance is on the rise. This resistance is mediated by the mutations in the domain V of 23S rRNA, presence of the cfr gene, or the mutations in the ribosomal proteins.^[4,8,9] There are only very few Indian studies describing the mechanism of resistance to linezolid in Staphylococcus haemolyticus. Resistance mediated by cfr and mutation in 23S rRNA have been reported with G2576T mutation as the most common.^[6,9]

This study was undertaken to detect and characterize resistance to linezolid among clinical isolates of Staphylococcus haemolyticus.

Materials and Methods

Molecular Methods

Results

Among the 84 isolates, the nuc gene was present in all the isolates, confirming the identification as Staphylococcus haemolyticus. The resistance exhibited to various classes of antimicrobials is as follows: ampicillin 95.2% (80/84), cefuroxime 79% (66/84), cefoxitin 79% (66/84), cefotaxime 79% (66/84), erythromycin 88% (74/84), clindamycin 57% (48/84), ciprofloxacin 72.6% (61/84), and linezolid 3.5% (3/84). All isolates were susceptible to vancomycin and teicoplanin.

Methicillin resistance was detected by oxacillin and cefoxitin disc diffusion method. Resistance to oxacillin was observed in 67/84 and resistance to cefoxitin was detected in 66/84 of the study isolates. The mecA gene was present in 98.5% (66/67) of the oxacillin-resistant isolates.

The linezolid-resistant isolates (n = 3) had MIC of greater than 128 µg/mL. Cfr gene was detected in all the three linezolid-resistant isolates. The domain V of the 23S rRNA gene was amplified by PCR. The obtained sequences were compared to the reference 23S rRNA gene sequences of Staphylococcus haemolyticus (JCSC1435). The BLAST alignment revealed G2603T point mutation in the domain V of 23S rRNA gene in two of the linezolid-resistant isolates.

The clinical details of the patient from whom LRSH was isolated are tabulated in ►Table 2.

Discussion

Staphylococcus haemolyticus is increasingly recognized as an important pathogen due to its ability to develop multiple drug resistance, its adaptability and ability to survive in the hospital environment, especially on medical devices.^[3] Linezolid, an oxazolidinone, is indicated for the treatment for a variety of Gram-positive infections; it is often the last-resort antibiotic for the management of infections caused by methicillin-resistant Staphylococci.^[4]

Resistance to linezolid in Staphylococci is due to one or more of the following mechanisms: acquisition of the cfr (chloramphenicol florfenicol resistance) gene, mutation in the central loop of domain V of the 23S rRNA, and mutation in the rplC and rplD genes, which encodes for the 50S ribosomal proteins L3 and L4, respectively.^[4,8,9] Transferable plasmid mediated cfr gene encodes for a ribosomal methyltransferase conferring resistance to phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A (PhLOPS_A).^[9,13] Since it confers resistance to other classes of antimicrobial agents, attention should be paid to the fact that linezolid-resistant strains might be selected during treatment with any of these drugs.^[14] Co-occurrence of cfr mediated resistance and mutational resistance has also been documented more frequently.^[4,6,13] The ability of cfr gene to be transmitted between different bacterial strains and species is a cause for concern.

In this study, linezolid resistance was detected in three isolates and their MIC was greater than 128 µg/mL. All the three isolates carried the cfr gene and among them the mutation in the domain V of 23S rRNA was detected in two isolates. The BLAST alignment revealed a novel G2603T point mutation in the domain V of 23S rRNA gene in two of the linezolid-resistant isolates. The G2603T mutation has not been reported previously in India. The G2603T mutation has been reported in China among Staphylococcus epidermidis, Staphylococcus capitis^[4,8] and in Brazil among Staphylococcus hominis, Staphylococcus epidermidis, and Staphylococcus haemolyticus.^[15,16] Other mutations in domain V of 23S rRNA reported in literature include G2614T, C2384T, T2500A, C2534T, T2504T, G2447T, G2215A, C2190T, C2505A, and G2631T among other clinical Staphylococci.^[9,13,15,16] However, none of the above mutations was observed in this study. ►Table 3 represents the mutations reported in the domain V of 23S rRNA encoding for linezolid resistance in Staphylococcus haemolyticus since 2012.^{[5,6,13,15,17-22]}

Following its first detection in 2001, sporadic cases of linezolid resistance have been reported globally. In India, the first case report on LRSH from North India was published in 2011^[23] followed by another report in 2012^[24] and later from South India.^[6] In the former, mechanism of resistance was not studied, while the latter described the mechanism of resistance was due to the presence of cfr and mutation in domain V of 23S rRNA. A recent study published from South India reported linezolid resistance in 3.7% (13/356) of Staphylococcus haemolyticus with 12 isolates harboring the cfr gene. Mutation in domain V of 23S rRNA was not looked for in their study.^[3] In a hospital from Delhi, nine LRSH isolates were characterized and it was found that all of them carried the cfr gene along with mutation G2614T in domain V of 23S rRNA.^[13]

A study from Vietnam carried out whole genome sequencing and demonstrated the transferability of the plasmid carrying the cfr gene.^[14] However, in this study gene transfer experiments were not carried out.

This study reports dual mechanisms of resistance to linezolid. Although the mutational resistance to linezolid poses a threat in clinical practice, the acquisition of the cfr gene is threatening because of its potency for horizontal transmission between species. Only one isolate carried the cfr gene alone and lacked any mutations, which is similar to the observations made in previous studies.^[2,13]

In this study, of the three patients who had infection with LRSH, two patients underwent wound debridement and ray amputation for removal of nidus of infection and source control. Though these patients were treated with beta lactam antibiotics and fluoroquinolone, they recovered and were discharged from the hospital. It may be reasonably assumed that Staphylococcus haemolyticus could have been a colonizer in the wound and the recovery may be attributed to source control. Follow-up was lost in one patient.

Since many CONS are usually considered as part of normal skin flora, most clinical laboratories do not test for antimicrobial susceptibility unless from a sterile site such as blood. These organisms have relatively low virulence but are now increasingly recognized as clinically significant. As the pathogenic significance becomes apparent, it becomes necessary to characterize them and study their antimicrobial susceptibility profile.^[14]

Conclusion

The presence of cfr gene along with mutations is alarming. Prudent use of linezolid and strengthening implementation of infection control measures and screening of patients with linezolid resistant-CONS should be mandated to curtail the spread of resistance and preserve the drug.