Inducible Clindamycin Resistance in Staphylococcus aureus Isolated from Nursing and Pharmacy Students

INTRODUCTION

Clindamycin, an antimicrobial belonging to the macrolide-lincosamide-streptogramin B (MLS _B) family, is frequently used for treatment of skin and soft tissue infections caused by Staphylococcus aureus (S. aureus). However, the widespread use of the MLS _B family of antimicrobials has led to the emergence of resistance. ^[1] The common mechanism of resistance is mediated by erm genes that encode enzymes conferring inducible (MLS _B i) or constitutive (MLS _B c) resistance to MLS _B agents by reducing binding by these agents to the bacterial ribosome. ^[2-4]

Isolates with inducible clindamycin resistance are found to be resistant to erythromycin but susceptible to clindamycin when these discs are not placed adjacent to each other during antimicrobial sensitivity testing. Consequently, laboratory identification of such isolates is often missed, resulting in inappropriate therapeutic use of clindamycin and treatment failure. ^[2] These isolates can be detected by the D-test, a disc diffusion test in which induction of clindamycin resistance by erythromycin is tested. ^[2,5,6]

S. aureus can colonize healthy community-dwelling individuals, who are not only at an increased risk for developing subsequent infections, but also transmit the pathogen to other individuals. ^[7-9] Methicillin-resistant S. aureus (MRSA) is an important nosocomial pathogen and healthcare professionals, who are known to be carriers of MRSA, can transmit the pathogen to patients under their care, thereby leading to various complications associated with MRSA infections (like pneumonia and septicemia) in the patients. ^[7]

In this study, the prevalence of inducible clindamycin resistance in S. aureus isolated from nursing students and pharmacy students (representing carriers exposed and not exposed to hospital environment, respectively) was evaluated, with particular regard to MRSA.

MATERIALS AND METHODS

The present study was conducted at the department of microbiology of our medical college from 30 June, 2010 to 31 August, 2010. Institutional ethical committee approval was obtained.

One hundred nineteen students of the age group 18-23 years of the college of nursing attached to our hospital were included in the study. All students attended rotating sessions for at least 4 h daily in various hospital departments for the last 6 months. They represented the group exposed to hospital environment.

One hundred age-matched students of the college of pharmacy in our city were also studied. None of the students in this group had a history of hospitalization or regular visits to a hospital in the last 6 months. They represented the group not exposed to hospital environment.

None of the subjects had a history of illness or treatment with an antibiotic in the last 6 months. An informed written consent was obtained from each of the subjects.

Nasal, throat, palmar, and web-space swabs were collected from each of the subjects. S. aureus was identified phenotypically by growth on mannitol salt agar, Gram's stain, catalase test, and slide and tube coagulase tests. ^[10-12] Antimicrobial susceptibility testing of the isolates to penicillin (10 U), cefoxitin (30 μg), erythromycin (15 μg), and clindamycin (2 μg) (HiMedia, Mumbai) was carried out by the standard disc diffusion test as per Clinical and Laboratory Standards Institute (CLSI) guidelines. ^[13] The erythromycin and clindamycin discs were placed 15 mm apart edge to edge. ^[2,5]

Isolates showing resistance to erythromycin (zone size ≤ 13 mm) and sensitivity to clindamycin (zone size ≥ 21 mm), as well as giving a D-shaped zone of inhibition around clindamycin with flattening adjacent to the erythromycin disc were considered to show inducible clindamycin resistance [Figure 1]. ^[5,6] S. aureus BAA 977 and S. aureus BAA 976 were used as the positive and negative controls, respectively.

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Figure 1:

D-test showing a blunted zone of inhibition around the clindamycin disc adjacent to the erythromycin disc

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MRSA was detected by the cefoxitin disc diffusion test, using a 30 μg disc (HiMedia, Mumbai) (an inhibition zone diameter of ≤ 21 mm was reported as oxacillin or methicillin resistant and a zone diameter of ≥ 22 mm was considered sensitive) and by growth on oxacillin screen agar, incorporating 4% NaCl and 6 μg/ml of oxacillin (HiMedia, Mumbai), as per CLSI guidelines. ^[12-14]

Quality control strains-MRSA ATCC 43300 and methicillin-sensitive S. aureus (MSSA) ATCC 25923-were used as positive and negative controls, respectively.

Association of exposure to hospital environment with carriage of S. aureus showing MLS _B i phenotype, and association between methicillin resistance and expressionof MLS _Bi phenotype were evaluated using the chi-square (χ²) test.

RESULTS

In the exposed group, 36 individuals were found to be carriers of S. aureus. Isolates from six (16.7%) individuals showed inducible clindamycin resistance. From the non-exposed group, 34 individuals were found to be carriers of S. aureus. Isolates from two (5.9%) individuals showed inducible clindamycin resistance.

The number of isolates showing MLS _B i and MS (resistance to erythromycin but sensitivity to clindamycin without forming a D-zone around the clindamycin disc) phenotypes in the groups exposed and not exposed to a hospital environment, as well as their categorization as MRSA and MSSA, is shown in [Table 1].

None of the isolates showed constitutive clindamycin resistance (MLS _B c phenotype).

The percentage of inducible clindamycin resistance was higher among the group exposed to a hospital environment than among the group not exposed. However, this was not found to be statistically significant (χ² = 2.02; P > 0.05).

The percentage of inducible clindamycin resistance was also higher among MRSA isolates compared to MSSA isolates [Table 2] and this was found to be statistically significant (χ² = 6.38; P < 0.05).

Of the 36 S. aureus isolates obtained from the group exposed to hospital environment, 13 were resistant to erythromycin; of the 34 S. aureus isolates obtained from the non-exposed group, 7 were resistant to erythromycin.

The distribution of S. aureus isolated from different carriage sites is given in Table 3.

DISCUSSION

Clindamycin provides an appropriate alternative for treatment of infections caused by isolates of S. aureus that are resistant to other antimicrobials like β-lactams, fluoroquinolones, and macrolides. However, isolates that are resistant to erythromycin may also show inducible resistance to clindamycin which can lead to treatment failure.

In our study, 11.4% (8 out of 70 individuals in both the study groups taken together) were colonized with S. aureus isolates showing the MLS _B i phenotype. Other Indian studies on clinical isolates of S. aureus showed MLS _B i prevalence of 14.5% and 13.1%. ^[5,15] Results of studies in other countries varied considerably with MLS _B i prevalence of 25.4% and 52% being reported among S. aureus. ^[2,16]

The group exposed to a hospital environment had a larger percentage (16.7%) of individuals colonized with S. aureus isolates with the MLS _B i phenotype compared to the group not exposed (5.9%) [Table 1]. This could be attributed to the fact that antibiotic resistance is more likely to emerge among S. aureus isolated from a hospital due to widespread antibiotic use and the resultant selection pressure created in a hospital environment. However, we found that the association between exposure to hospital environment and acquisition of isolates with the MLS _B i phenotype was not statistically significant. This could be attributed to the limited number of isolates with the MLS _B i phenotype that we obtained in our study.

More MRSA isolates showed MLS _B i phenotype (27.8%) compared to MSSA isolates (5.8%) [Table 2]. Our results vary a little compared to other Indian studies that reported MLS _B i prevalence of 27.6% and 38.4% among MRSA, and 1.6% and 12.9% among MSSA. ^[5,15] Association of methicillin resistance with the presence of the MLS _B i phenotype was found to be statistically significant in our study.

It was found that in the exposed group, most isolates showing the MS phenotype were also resistant to methicillin, whereas in the non-exposed group, all isolates showing MS phenotype were sensitive to methicillin [Table 1].

In conclusion, it is very likely that isolates of S. aureus resistant to β-lactams will also show inducible clindamycin resistance. Exposure to hospital environment is not found to be a risk factor for carriage of S. aureus with the MLS _B i phenotype. However, given that (to the best of our knowledge), this is the first study to explore the prevalence of the MLS _B i phenotype in S. aureus isolated from carriers, further evaluation with a larger sample size is required for confirmation of the existence of an association between exposure to hospital environment and carriage of S. aureus with the MLS _B i phenotype.