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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 2  |  Page : 105-109

Detection of inducible clindamycin resistance among staphylococcus isolated from patients attending a tertiary care hospital in South India


1 Department of Microbiology, Sri Venkateswara Medical College, Tirupati, Andhra Pradesh, India
2 2Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India

Date of Submission22-Jun-2019
Date of Decision02-Apr-2020
Date of Acceptance03-Apr-2020
Date of Web Publication4-Aug-2020

Correspondence Address:
Saranya Mallamgunta
Department of Microbiology, Sri Venkateswara Medical College, Tirupati 517 507, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JCSR.JCSR_86_19

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  Abstract 


Background: Resistance to antimicrobial agents led to the usage of macrolide-lincosamide-streptogramin B (MLSB) family to treat staphylococcal infection.
Methods: One hundred and ninety one isolates obtained from various clinical samples such as pus, wound, swab, blood urine, and other body fluids were processed, and staphylococci were isolated as per standard bacteriological techniques. The antibiotic sensitivity was tested by Kirby–Bauer disc-diffusion method.
Results: Out of 191 isolates, 32% were coagulase-negative staphylococci (CoNS). Of these, 48% and 65% were resistant to erythromycin, respectively. Among erythromycin-resistant S. aureus, 30% each showed constitutive resistance and inducible clindamycin (CL) resistance, and 60% showed MS phenotypes by the D-test. Of the CoNS, 18% showed inducible CL resistance. Among 130 S. aureus isolated, 15% each had constitutive macrolide-lincosamide-streptogramin B resistance (MLSBc), inducible macrolide-lincosamide-streptogramin B resistance (MLSBi) and 25 (19%) were D test-negative, were resistant to macrolides, streptogramins, sensitive to lincosamides. Among 61 CoNS isolated 9 (15%) were MLSBc resistant, 7 (11%) were MLSBi resistant and 24 (39%) were D-test negative (MS phenotype). Resistance MLSBi percentage was more among S. aureus 15% compared to CoNS 11%.
Conclusions: The ER-CL disc-approximation test (D-test) emerged as a simple, auxiliary, easy to perform, reliable method and can be used as routine laboratory method for detecting inducible, constitutive and MS phenotypes.

Keywords: D-test, inducible clindamycin resistance, methicillin resistance


How to cite this article:
Mallamgunta S, Vasudeva Naidu K H, Ramakrishna N. Detection of inducible clindamycin resistance among staphylococcus isolated from patients attending a tertiary care hospital in South India. J Clin Sci Res 2020;9:105-9

How to cite this URL:
Mallamgunta S, Vasudeva Naidu K H, Ramakrishna N. Detection of inducible clindamycin resistance among staphylococcus isolated from patients attending a tertiary care hospital in South India. J Clin Sci Res [serial online] 2020 [cited 2020 Oct 31];9:105-9. Available from: https://www.jcsr.co.in/text.asp?2020/9/2/105/291380




  Introduction Top


Staphylococcus aureus is one of the most common human pathogens capable of causing a wide range of infections.[1] The resistance to antimicrobial agents among staphylococci is an increasing problem. Penicillin-resistant staphylococcus strains began emerging shortly after the introduction of penicillin in the medicine in 1940. Today, the percentage of penicillin-resistant strains has risen to 75%–95%. Most penicillin-resistant staphylococcal strains produce beta-lactamase which hydrolyses the b-lactam ring of the antibiotic.[2] Clindamycin (CL) can be used for the treatment of staphylococcal infections. CL is a useful choice in case of penicillin allergy.[3]

CL is a lincosamide antibiotic, developed in 1966 by chemically modifying the naturally occurring lincomycin. CL belongs to the macrolide-lincosamide-streptogramin B (MLSB) family. Its spectrum of activity includes staphylococci, streptococci, most anaerobic bacteria, Chlamydia and certain protozoa. The wide spread use of the MLSB family of antimicrobials has led to the emergence of resistance.[4] CL represents a useful option for therapy of various staphylococcal infections. However, clinical failure of CL therapy was documented for some of the strains of CL sensitive but erythromycin-resistant phenotypes.[5] These failures were due to inducible resistance to CL. In such cases, routine antibiotic susceptibility test cannot identify these strains. The D-test is employed to detect these strains.

The purpose of our study was to determine the sensitivity pattern of staphylococcal isolates from infected patients to common antibiotics and to detect inducible CL resistance among staphylococcal isolates from patients attending a tertiary care hospital in South India.


  Material and Methods Top


The present study was carried out in the Department of Microbiology, Sri Venkateswara Medical College, Tirupati, India, after obtaining the Ethical Committee Clearance. One hundred and ninety one isolates from various clinical samples obtained from patients who were admitted in the Sri Venkateswara Ramnarain Ruia (SVRR) Government General Hospital, the tertiary care teaching hospital attached to Sri Venkateswara Medical College, Tirupati were included in the study.

The samples were inoculated on nutrient agar, blood agar and Mac Conkey agar. The inoculated plates were incubated at 37 °C overnight. If any growth was seen on the plates, it was processed according to the standard bacteriological techniques.[6] Preliminary tests were followed by both slide and tube coagulase tests and other biochemical reactions such as phosphatase test, mannitol fermentation and urease test for the identification of S. aureus and coagulase-negative staphylococci (CoNS).

The antibiotic sensitivity pattern of isolated S. aureus and CoNS was done by Kirby–Bauer disc-diffusion method as per the CLSI guidelines.[7] The following antibiotics were used: co-amoxyclav (30 μg); cefoxitin (30 μg), ceftazidime (30 μg), penicillin (10 units), erythromycin (15 μg), and CL (2 μg).

Methicillin resistance was detected by using cefoxitin disc as it is more sensitive than oxacillin for routine laboratory detection of methicillin-resistant S. aureus (MRSA) isolates in clinical settings.[8]

Lawn cultures of the isolates were made on Muller-Hinton agar using suspension of each isolate matching 0.5 McFarlands turbidity. CL and erythromycin disks containing 2 μg and 15 μg of each were placed with 15-mm gap between the edges.

Inducible resistance was defined as blunting of the clear zone of CL on the side adjacent to erythromycin, producing a D-shaped zone denoting inducible CL resistance (D-test positive). The absence of a blunted zone of inhibition (circular zone) (D-test negative), suggested that the test strain is truly susceptible to CL as per CLSI guidelines.[7] The following definitios were used erythromycin sensitive ≥23 mm, erythromycin resistant ≤13 mm, CL sensitive ≥21 mm, CL resistant ≤14 mm.

Three different phenotypes were appreciated after testing and interpreted as follows: (i) MS phenotype: Staphylococcal isolates exhibiting resistance to erythromycin (zone size <13 mm) while sensitive to CL (zone size >21 mm) and giving circular zone of inhibition around CL disc was MS phenotype; (ii) inducible clindamycin-resistant phenotype (inducible MLSB [MLSBi]) – staphylococcal isolates showing resistance to erythromycin while being sensitive to CL giving D-shaped zone of inhibition around CL with flattening toward erythromycin disc (D-test positive); and (iiii) constitutive clindamycin-resistant phenotype (constitutive MLSB [MLSBc])– This phenotype showed resistance to both erythromycin and CL.


  Results Top


One hundred and ninety-one staphylococcal isolates were tested for antibiotic susceptibility testing by the disc-diffusion method. The staphylococcal isolates resistant to erythromycin were subjected to D-test for detecting inducible CL resistance. Forty-four per cent of isolates were obtained from blood followed by pus (25%), urine (20%), conjunctival swab (4%), throat swab (3%) and sputum (2%). Sixty eight per cent of staphylococcal isolates were coagulase-positive and 32% were coagulase-negative.

The antibiotic profile of staphylococcus tested on Muller-Hinton agar, incubated at 37 °C for 24 h, showed that, among 191 isolates, maximum resistance is seen to penicillin (98%) followed by amoxyclav (73%). Minimum resistance is seen to CL (28%). Among S. aureus, 63% were methicillin resistant, and among CoNS, 53% were methicillin resistant. Methicillin resistance percentage was more in S. aureus. Forty-three per cent of study isolates were MRSA, 25% were MSSA, 17% were MRCONS and 15% were MSCONS. Hence, maximum isolates obtained were MRSA followed by MSSA, MRCONS and MSCONS. MRSA were obtained more from blood followed by pus.

Among S. aureus, 48% were erythromycin resistant and among CoNS 65% were erythromycin resistant. The erythromycin-resistant strains of S. aureus and CoNS were subjected to D-test to detect MLSBc, MLSBi and MS phenotypes. Among erythromycin-resistant S. aureus, 30% showed constitutive resistance, 30% showed inducible CL resistance and 40% showed MS phenotypes by D test. Among 130 S. aureus isolated, 19 (15%) were MLSBc, 19 (15%) were MLSBi and 25 (19%) were MS phenotypes and among 61 CoNS isolated 9 (15%) were MLSBc, 7 (11%) were MLSBi and 24 (39%) were MS phenotype [Table 1].
Table 1: Phenotypic distribution among Staphylococcus aureus and coagulase-negative staphylococci

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In CoNS 18% of strains which were erythromycin resistant shows inducible CL resistance. Among 130 S. aureus isolated, 19 (15%) were MLSBc, 19 (15%) were MLSBi and 25 (19%) were MS phenotypes and among 61 CoNS isolated 9 (15%) were MLSBc, 7 (11%) were MLSBi and 24 (39%) were MS phenotype.

Among MRSA inducible CL resistance was 16% (13/82). Among MSSA, 12% (6/48) was MLSBi. Among MRCoNS 12% was (7/61) MLSBi. Among MSCONS, 10% was MLSBi. Proportion of MLSBi was more among S. aureus (15%) compared to CoNS (11%).

Comparison of our observations with observations from other studies is shown in Tables 2,[9],[11],[15] 3,[9],[14],[16],[17],[18],[19] and 4.[5],[11],[20],[21]


  Discussion Top


S. aureus and CoNS are the important causes of nosocomial and community-acquired infections. S. aureus can cause a range of illnesses, from minor skin infections, such as pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, and abscess, and diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic-shock syndrome, bacteraemia and sepsis. CoNS now represent one of the major nosocomial pathogens, with S. epidermidis and S. haemolyticus being the most significant species.

The emergence of drug resistance among staphylococci is an increasing problem.[9] MRSA is a notorious nosocomial pathogen, and its rate has dramatically increased in the recent years. The increasing frequency of the infections with MRSA, and the changing drug-susceptibility patterns have led to a renewed interest in the use of MLSB antibiotics to treat such infections, CL a lincocamide being the preferred agent due to its excellent pharmacokinetic properties.[9]

Of 191 study isolates, maximum isolates were obtained from blood 85 (44%), followed by pus 47 (25%) and urine 39 (20%). These observation correlates with the study done in Nepal[10] where out of 306 isolates of staphylococcus 120 (40%) were obtained from blood followed by pus 83 (27%) and urine 63 (20%). Maximum isolates were obtained from 0 to 10 years' age group. This is similar to a study conducted in Nepal[10] where maximum isolates were obtained from 0 to 10 years' age group. Among staphylococci, 68% (130/191) were coagulase positive and 32% (61/191) were coagulase negative. Similar observations were reported in another study[9] 64% (239/373) isolates were coagulase positive. In another study[11] 76% (259/337) solates were coagulase positive. Among 61 CoNS, 38 were isolated from blood.

The present study demonstrated that overall rates of resistance to commonly prescribed antibiotics in staphylococcal isolates were increased. Among S. aureus isolates, 97.3% were resistant to penicillin and 28% of isolates were resistant to CL. Similar observations were reported in another study[10] where 95% of isolates were resistant to penicillin and 28% of isolates were resistant to CL.

In the present study, among S. aureus isolates, 63% (82/130) were methicillin-resistant strains. Our observations are similar to that reported in another study[12] where 71% of isolates were resistant to methicillin.

In the present among CoNS, 53% (32/61) were methicillin resistant. This is similar to a study conducted in 2013[13] where 65% of CoNS were methicillin resistant. We observed that maximum isolates of MRSA were obtained from blood followed by pus; Similar observations were reported in another study.[9] In our study, maximum isolates of MRSA were obtained from 0 to 10 years of age. In the present study, 48% (63/130) of S. aureus were erythromycin resistant and 52% (67/130) were erythromycin sensitive; this is similar to studies conducted in 2013[12] where 48.3% of S. aureus were resistant to erythromycin, and other study conducted in 2012[11] where 50% of S. aureus were resistant to erythromycin.

In our study, CoNS showed 65% (40/61) resistance to erythromycin which is very high compared to another study[11] where only 51% of CoNS showed resistance to erythromycin.

Among erythromycin-resistant S. aureus isolates, 30% (19/63) had constitutive resistance, 30%(19/63) expressed inducible resistance and 40% (25/63) had MS phenotype, this is similar to study conducted in 2013[13] where 33% of erythromycin-resistant S. aureus had constitutive resistance, 34% expressed inducible resistance and 32% had MS phenotype [Table 1].

Among erythromycin-resistant CoNS, 23% (9/40) had constitutive resistance, 18% (7/40) expressed inducible CL resistance and 59% (24/40) had MS phenotype; this is similar to a study conducted 2012[11] where 23% had constitutive resistance, 15% expressed inducible resistance and 62% had MS phenotype [Table 2]. Among staphylococcal isolates, 14%(26/191) expressed inducible CL resistance, 15% (28/191) had constitutive CL resistance and 25% (49/191) had MS phenotype. Among S. aureus isolates, 15% (19/130) had constitutive resistance, 15% (19/130) expressed inducible resistance and 19% (25/130) had MS phenotype. Similar observations were reported in another study[11] where 10% of S. aureus had constitutive resistance, 11% expressed inducible resistance and 30% had MS phenotype. Inducible CL resistance among MRSA was 16% (13/82). Similar results were documented in another study[9] where it was reported in 19% of the MRSA. Among MSSA, 12% (6/48) was MLSBi. Similar observations were reported in a study[14] from Tamil Nadu where 11% of the MSSA was MLSBi. Among MRCONS, 13% showed inducible resistance, and among MSCONS, 10% showed inducible resistance [Table 3].
Table 2: Comparison of erythromycin resistance pattern and different phenotypes in the present stud with other published studies[9],[11],[15]

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Table 3: Comparison of inducible macrolide lincosamide and Group B streptogramins among various staphylococcal strains in the present study with other published studies[9],[14],[16],[17],[18],[19],[20]

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Among MRSA, inducible CL resistance was 16% and constitutive CL resistance among MRSA was 15%, which is similar to other reports [Table 4].
Table 4: Comparison of phenotypes among methicillin-resistant Staphylococcus aureus in the present study with data published in other studies[5],[11],[20],[21]

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In our study, MS phenotype among 191 isolates was 49 (26%), and among MSSA, it was 17% (8/48), MRCONS 47% (15/32), and MSCONS 31% (9/29). Similar results were reported in another study,[15] where, Among MRSA, 21% (17/82) had MS phenotype.

Among pus samples, MLSBi was 19% (9/47) which is higher compared to other samples. MLSB resistance is the most widespread and the clinically important mechanism of resistance among the Gram-positive organisms due to the production of methylases and efflux proteins. The emergence of the resistance to multiple antibiotics among the Gram-positive organisms has left limited options for the clinicians and an appropriate. The above findings suggest that the MLSBi phenotype widely varies, this depends on the basis of the geographical location, hospital environment, patient age, clinical samples which are examined, bacterial species involved and the antibiotic susceptibility profile of the bacteria.[16],[17],[18],[19],[20] The frequency of MLSBi ranges from 7%–94%.[21] The CL therapeutic decision is not possible with the routine antibiotic susceptibility data. The D-test becomes necessary to identify the inducible CL resistance.

In our present study, percentage of inducible clindamycin resistance among staphylococcal isolates was 14%, which is almost equal to constitutive clindamycin resistance i.e., 15%. The increasing prevalence of the inducible resistance as compared to that of the constitutive resistance among staphylococci suggests the importance of identifying MLSBi phenotypes.

CL can be omitted in patients with infections which are caused by strains with MLSBi phenotype, to avoid possible therapeutic failures. On the other hand, negative result for inducible CL resistance confirms CL susceptibility and provides a very good therapeutic option. Detection of inducible CL resistance can be missed by routinein vitro susceptibility tests, to detect MLSBi ER-CL disc-approximation test or the D-test is needed, the test was simple, auxiliary, easy to perform, reliable method and can be used as routine laboratory method for detecting inducible, constitutive and MS phenotypes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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