Phenotypic characterization and Molecular detection of Inducible and Constitutive Clindamycin resistance among Staphylococcus aureus isolates in a Tertiary Care Hospital

 

G. Mahalakshmi¹, Dr. P. Neelusree²*, Dr. M. Kalyani³

¹Principle, Investigator M.Sc., Medical Microbiology, Saveetha Medical College and Hospital,

Saveetha Institute of Medical and Technical Sciences, Chennai - 602105.

²MD Associate Professor, Saveetha Medical College and Hospital, Saveetha Institute of Medical and Technical Sciences, Chennai - 602105.

³Head of the Department, Saveetha Medical College and Hospital, Saveetha Institute of Medical and Technical Sciences, Chennai - 602105.

 

ABSTRACT:

Background: Staphylococcus aureus is Gram positive cocci. The pyogenic bacteria which is responsible for a variety of diseases that ranges in severity from mild skin and soft tissue infections to life-threatening conditions such as endocarditis, pneumonia, and sepsis. There is a scenario of increasing Methicillin-resistant Staphylococcus aureus (MRSA) infections, the macrolide-lincosamide-streptogramin B (MLSB) group of antibiotics they have different structure with same mechanism of action which serves as one good alternative. There is a frequency of increasing Methicillin Resistant Staphylococcus aureus (MRSA) infections and their change in antimicrobial resistance pattern. There is a concern about use of this antibiotic in the presence of Erythromycin resistance because of the possibility of inducible resistance among the members of Macrolide, lincosamide, Strepto-gramin B (MLSB) group. The invitro resistance exhibited by Staphylococcus aureus to erythromycin, Clindamycin, and other drugs of MLSB groups is due to the expression of ribosomal methylases(erm) genes. The detection of inducible Clindamycin resistance can limit the effectiveness of these drugs. Objective of the study: To isolate of Staphylococcus aureus from various clinical samples to differentiate between Methicillin resistant Staphylococcus aureus (MRSA) and Methicillin sensitive Staphylococcus aureus (MSSA) by conventional methods. To detect inducible and constitutive Clindamycin resistance in Staphylococcus aureus isolates by D test. To detect ermA gene responsible for resistance by PCR. Methodology: This cross sectional study was done for a period of six months. Totally 106 Staphylococcus aureus isolates was obtained various clinical samples were processed using standard guidelines. Result: From the 106 isolates of Staphylococcus aureus 67(63.3%) were MSSA and 39(36.7%) were MRSA. D-test was positive in n=9 of the n=21 MRSA and n=17 of the n=85 MSSA, which denotes inducible Clindamycin resistance. N- 9 of MRSA and n=13(22%) of MSSA showed Constitutional Clindamycin resistance. The statistics show that there is a significant Difference in constitutive resistance between MRSA and MSSA. In India ermA gene is most prevalent, out of 22 d-test positive n=13 ermA gene were detected (n=3-MRSA and n=10-MSSA) by using conventional PCR. Conclusion: The MLSB family of antibiotics is one such alternative and CD is preferred. Clinical microbiology laboratories should report inducible Clindamycin resistance in Staphylococcus aureus and D-test can be used as a simple, auxiliary and reliable method to Delineate inducible and constitutive Clindamycin resistance in routine clinical laboratories.

 

 

INTRODUCTION:

Staphylococcus aureus is Gram-positive bacteria, with diameters of 0.5–1.5μm and characterized by individual cocci, which divide in more than one plane to form grape-like clusters. They are non-motile, non-spore forming facultative anaerobes that grow by aerobic respiration or by fermentation they are ubiquitous, most common significant pathogens causing nosocomial and community-acquired infections worldwide. This is a pyogenic bacteria which is responsible for a variety of diseases that ranges in severity from mild skin and soft tissue infections to life-threatening conditions such as endocarditis, pneumonia, and sepsis. This can further spread and results in various clinical manifestations of localized infections such as carbuncle, cellulitis, impetigo, bullosa or wound infection. The organism can enter into blood and spread systemically to different organs causing sepsis. This haematogenous spread may result in endocarditis, osteomyelitis, renal carbuncle, septic arthritis and epidural abscess. Without a blood stream infection, specific syndromes can occur due to extra cellular toxins of S. aureu¹.

 

There is a scenario of increasing Methicillin-resistant Staphylococcus aureus (MRSA) infections, the macrolide-lincosamide-streptogramin B (MLSB) group of antibiotics; they have different structure with same mechanism of action which serves as one good alternative². The Methicillin resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial and community acquired infections. There is a frequency of increasing Methicillin Resistant Staphylococcus aureus (MRSA) infections and their change in antimicrobial resistance pattern³.

 

The antibiotics of MLSB family have three different mechanisms of drug resistance– target site modification, enzymatic antibiotic inactivation and Macrolide efflux pumps Clindamycin, is the limited choice of antimicrobials effective against MRSA. There is a concern about use of this antibiotic in the presence of Erythromycin resistance because of the possibility of inducible resistance among the members of Macrolide, Lincosamide, Strepto-gramin B (MLSB) group. The phenotype of Staphylococcus aureus can be either constitutive MLSB (cMLSB) or inducible MLSB (iMLSB)³.

 

The in-vitro resistance exhibited by Staphylococcus aureus to erythromycin, Clindamycin, and other drugs of MLSB groups is due to the expression of ribosomal methylases (erm) genes.

 

This resistance is referred to as the cMLSB phenotype. However, in some Staphylococci the erm gene which synthesize methylase enzyme, induces the Clindamycin resistance it is referred to as iMLSB phenotype. Hence the organism is resistant to erythromycin and falsely susceptible to Clindamycin in-vitro. Some methods like- broth micro dilution testing, automated susceptibility testing devices, or Epsilometer test which do not detect the inducible Clindamycin resistance. Reporting of falsely susceptible Clindamycin will lose its effectiveness in vivo and thereby increases the chance of therapeutic failures².

 

The detection of inducible Clindamycin resistance can limit the effectiveness of these drugs. The demonstration of inducible MLSB phenotype among Staphylococcus aureus isolates which are susceptible to Clindamycin and resistant to Erythromycin can be detected by Double Disk diffusion approximation test or D – test. This test is mainly based on the placement of erythromycin disk adjacently to the Clindamycin with optimal spacing. As the erythromycin diffuses through the agar, resistance to the lincosamide is induced, which results in a flattening or blunting of the lincosamide zone of inhibition adjacent to the erythromycin disk, giving a D shaped zone (D-zone effect). Failure in identification of inducible MLSB resistance may lead to therapeutic failure in clinical cases. Labeling of all erythromycin resistance staphylococci as Clindamycin resistance prevents the use of Clindamycin drug. Strains with constitutive MLSB resistance (cMLSB) shows in vitro resistance to all of these agents- the alternative drugs given to penicillin-allergic patients were MLSB antibiotics. Low levels of erythromycin are the most effective inducer of inducible MLSB resistance.

 

Clindamycin is available as oral tablets (150 and 300 mg), parenteral injection (intramuscular or intravenous), and topical and vaginal formulations. Usual parenteral doses are 600mg every 6 to 8 h to 900mg every 8 h. Typical oral doses are 150 to 450mg qid. Topical formulations include a 2% ointment and 2% vaginal gel. The genotyping of medically important bacteria was mean while described in a large number of reports, especially for arbitrarily

 

amplifying variable regions of the bacterial genomic DNA. It helps in identification of possible epidemic strains which in spread by migration of patients, the same suitable typing system use have a greater importance. A PCR-based typing system would be most appropriate due to its ease and speed of performance¹⁴.

 

The enzyme encodes erm gene which is responsible for inducible or constitutive resistance to MLSB agents via methylation of the 23S rRNA, educing binding by MLSB agents to the ribosome. Resistance is induced by the binding of a Macrolide to upstream translational attenuator sequences, it leads to changes of mRNA secondary structure, exposure to the ribosomal binding site and erm methylase translation takes place. All these alterations takes place in the 5_ upstream sequence which includes deletions, duplications and other mutations, it leads to constitutive MLSB resistance and constitutive expression of methylase gene.

In this study, the reliability of placing erythromycin and Clindamycin disks adjacently, where the standard disk dispenser method is compared to that of special disk approximation test that employs spacing the disc closer. The double disk diffusion approximation test is to predict the resistance among genotype was determined by molecular method by performing PCR for the erm A genes of Staphylococcus aureus from the clinical isolates.

 

Studies on the prevalence of drug resistant Staphylococcus aureus has not been done in our hospital. The data emerging out of this study helps in understanding the dynamics of this infection and provide inputs for antibiotic policy in the treatment of such infections.

 

MATERIALS AND METHODS:

This cross sectional study was carried out for a period of six months in the clinical Microbiology laboratory in Saveetha Medical College and Hospital, Thandalam- Chennai.

 

Samples like pus, wound swab, blood, other fluids collected were processed as per standard guidelines and Staphylococcus aureus isolates isolated from those samples were included for this study⁵.

 

Antimicrobial susceptibility test (Kirby Bauer disk diffusion method):

Antibiotic susceptibility testing was done on Muller Hinton agar plate as per standard guidelines and procedure. All the Staphylococcus aureus isolates that were resistant to erythromycin were tested for inducible Clindamycin resistance by double disk approximation test (D-test) as per CLSI guidelines. In this test, turbidity of Staphylococcus aureus is compared with 1% McFarland's standard. The suspension was lawned over the MHA plate and the antibiotic disc was placed over the lawned plated. An erythromycin disk (15μg) and Clindamycin (2μg) were placed 15mm apart edge-to-edge on MHA plate. Methicillin resistance was identified by placing Cefoxitin (30μg) disc on MHA plate and interpreted as per CLSI guidelines. Plates were analyzed after overnight of incubation at 37°C. On next day the plates were examined for the zone of inhibition around the antibiotic disc. The diameter of the inhibitory zone includes the diameter of the disc.

 

Interpretation:

Screening for Methicillin resistance Staphylococcus aureus mentioned in (Table1)

 

Table 1: Interpretation of Methicillin resistance

	Zone diameter breakpoint in mm
	Susceptible	Intermediate	Resistant
Cefoxitin	≥ 22	-	≤ 21

 

iMLSB phenotype: Isolates resistant to erythromycin but sensitive to clindamycin showing flattening of the zone of inhibition around clindamycin producing a “D” shaped blunting toward erythromycin disk (D-test positive). The organism is interpreted as clindamycin resistant (figure 1)

 

Figure 1: MRSA (D-Test positive)

 

cMLSB phenotype: Isolates resistant to both erythromycin and clindamycin with no or small zone of inhibition around clindamycin (Figure 2)

 

Figure 2: MRSA (D-Test Negative)

 

Methicillin-sensitive (MS) phenotype: Isolates resistant to erythromycin but sensitive to with a circular zone of inhibition around clindamycin (D-test negative) (Figure 3)

 

The organism is interpreted as clindamycin sensitive mentioned in Table: 2

 

Figure 3: MS Phenotype

 

Table 2: Interpretation of phenotypes of E and CD

Phenotype	E	CD	D test Result	Character of Phenotype
E and CD Susceptible	S	S
MS Phenotype	R zone size ≤13 mm	S zone size ≥ 21mm	Negative	Circular zone of Inhibition around CD
Inducible Phenotype	R zone size ≤ 13 mm	S zone size ≥ 21 mm	Positive	D Shaped zone of inhibition around CD with flattening towards E disc
Constitutive Phenotype	R zone size ≤ 13 mm	R zone size ≤14 mm

Molecular detection:

To amplify and to detect ermA gene in staphylococcus aureus⁹

Primers used for PCR: (Table 3)

 

Table 3:

Primer	Nucleotide sequence	Length of base pares
ermA Forward primer	5’- AAG CGG TAA ACC CCT CTG A- ‘3	190 bp
ermA Reverse primer	5’- TTC CGC ATT CCC TTC TCA AC- ‘3	190 bp

 

RESULT:

This Cross-sectional study conducted for a period of six months in Saveetha Medical College and Hospital. Total of 1610 clinical samples were analysed, among them totally 106 Staphylococcus aureus were isolated and included in this study.

 

Out of 106 samples studied, 59(55.6%) were male and 47(44.4%) were female. Among those 106 isolates 46 were from age group (21 to 40), 24 from age group (41 to 60), 20 from age group (61 to 80) and 16 from age group (0 to 20). Demographic details of the study subjects shown in (Table 4).

 

Table 4: Demographic details of Staphylococcus aureus

GENDER	0-20	21-40	41-60	61-80
MALE	9	25	13	12
FEMALE	7	21	11	8

 

Most of the Staphylococcus aureus isolates were obtained from Exudate 62(58.5%) samples followed by Blood 25(23.5%) and Respiratory 19(18%) (Figure4).

 

Figure 4: Isolation of staphylococcus aureus from various clinical samples

 

Among 106 isolates n=39(37%) were MRSA and n=67(63%) were MSSA. In our study methicillin sensitive is more predominant when compared to methicillin resistance as shown in figure-5.

 

Figure 5: Detection of MRSA and MSSA from various clinical samples

 

Statistical analysis:

D-test was positive in n= 9 of the 39 MRSA and n=13 of the n=67 MSSA, which denotes inducible Clindamycin resistance. n=18 of MRSA and n=9 of MSSA showed Constitutional Clindamycin resistance, n=4 of MRSA and n=12 of MSSA showed Ms Phenotype (Table 5).

 

Table 5: Distribution According to Inducible Clindamycin Resistant Phenotype (D-test) of MRSA and MSSA Isolates

	D-Test positive	D-Test negative	P value
MRSA (n=39)	9 (8.09) [0.1]	30 (30.91) [0.03]	0.652867
MSSA (n=67)	13 (13.91) [0.06]	54 (53.09) [0.02]	Not significant
Total	22	84

 

Distribution according to constitutive Clindamycin Resistant Phenotype of MRSA and MSSA Isolates

	Constitutive CD resistance	Not resistance	P value
MRSA (n=39)	18 -9.93 [6.55]	21 -29.07 [2.24]	0.000192	Significant
MSSA (n=67)	9 -17.07 [3.81]	58-49.93 [1.3]
Total	27	79

 

Distribution according to Ms phenotype of MRSA and MSSA

	Ms phenotype	Not showed Ms phenotype	P value
MRSA (n=39)	4 -5.89 [0.6]	35 -33.11 [0.11]	0.288449
MSSA (n=67)	12 (10.11) [0.35]	55 -56.89 [0.06]	Not significant
Total	16	90

 

Detection of ermA gene in inducible Clindamycin resistance Staphylococcus aureus:

In India ermA gene is most prevalent, out of n=22 D-test positive 13 ermA were detected (n=6-MRSA and n=7-MSSA) was detected by using conventional PCR (Figure 6).

 

Figure 6: Genotypic detection of ermA gene- Transilluminator under UV light

Lane: 1, 2, 3, 4, 6, 7, 9 and 12 – Detected; Lane: 5, 8, 13, 14 - Not Detected; Lane: M: Marker - 100bp; Lane: 10: Positive control -Detected;

Lane: 11: Negative control- Not Detected

 

DISCUSSION:

This study has demonstrated that careful examination of the shape of the Clindamycin zone adjacent to a standard 15-micro gram erythromycin disk in a conventional disk diffusion test can serve to detect S. aureus strains with inducible resistance to Clindamycin. Flattening of the Clindamycin disk diffusion zone in an erythromycin-resistant isolate (D-zone effect) appears to be a reliable indicator of iMLSB strains that harbor either the ermA gene. Constitutively MLSB-resistant strains are easily recognized by a Clindamycin zone diameter of 14mm with or without significant growth. Erythromycin susceptible strains do not possess inducible Clindamycin resistance.⁶

 

Isolation of S. aureus from Exudate samples in present study was 58.5% similar to a studies done by Priyanka Kalbhor et al⁶, isolation of S. aureus from pus and wound swab specimen was 55.35%, Mokta et al⁷, isolation of S. aureus from pus and wound swab specimen was 56%. Isolation of Staphylococcus aureus from pus in study by Adhikari et al⁸.

 

The wide spectrum of diseases caused by S. aureus includes skin and soft tissues infections, surgical site infections, infections of bones and joints. This might be reason for higher isolation of S. aureus from pus and wound swab samples. The knowledge of current antibiotic resistance pattern of S. aureus strains is necessary in selection of appropriate empirical treatment. In present study MSSA was found to be more predominant 63.3% and MRSA was found to be 36.7%. Similar prevalence rate of MRSA was obtained by other studies in India by Mokta et al⁸ (23.42%) and Gupta et al⁷ (25%). Deotale et al⁹ reported 49.8% MRSA in their study.

In this study, a total of 106 Exudate specimens were studied. Inducible phenotype was most common in pus/wound swab specimens with isolation rate of 21%. This is in concordance with study of Reddy et al¹⁰ who also reported inducible phenotype most commonly inpus specimen with isolation rate of 26.53%. However, Saffar et al¹¹ reported lower isolation rate of 4.16% of inducible phenotype in pus and wound swabs similarly in Priyanka kalbhor et al⁶ total of 176 pus/wound swab specimens Inducible phenotype was most common in pus/wound swab specimens with isolation rate of 28.41%.

 

Constitutive Clindamycin resistance in our study was observed in 7 (7%) of MRSA and 34 (32%) of MSSA7, which was similar to Subasini Majhi et al² Constitutive Clindamycin resistance was (8.7%) of MSSA and 30 (23.3%) of MRSA isolates.

 

In present study, Erythromycin and Clindamycin resistance was 60% respectively. Similarly, Shanthi et al¹² reported 62.5% and 35% resistance to Erythromycin and Clindamycin respectively. Deotale et al¹³ reported 32.39% and 18.22% of Erythromycin and Clindamycin resistant isolates respectively. MRSA is a major nosocomial and community pathogen causing significant morbidity and mortality. MRSA strains are important for their resistance to many other commonly used antibiotics. Vancomycin has been used to treat MRSA infections for more than three decades and there is an emergence of resistance to Vancomycin.¹⁴ In present study, MRSA was found to be 20.44%. However higher isolation rate was reported in some studies⁶.

 

The prevalence of erm genes may vary area to area and the population studied. The prevalence of ermA genes when compared to other studies. In our study (59%) 13 ermA out of 22 Inducible Clindamycin resistance were detected. In various studies prevalence of erm A was reported more than erm genes like Nizamiduran et al¹⁵., who reported 44 (52%) ermA genes¹⁶, Lim et al., reported 82.5% erm A and gene¹⁷. Similarly Gul et al., also reported high prevalence iof erm A genes i.e. erm A 55 (62%) respectively¹⁸.

 

Antimicrobial resistance in S. aureus has becoming an increasing problem among the both outpatients and inpatients of health care facilities. Clindamycin, a lincosamide, has always been an attractive opinion for MSSA and MRSA skin and soft tissue infections. It is available in oral and parenteral formulations, 90% oral bioavailability, less costly in comparison to newer drugs, good tissue penetration and inhibits the production of certain toxins and virulence factors in Staphylococci. However, resistance to Clindamycin is highly variable, and incidence of its resistant phenotypes varies by geographic regions and even between hospitals. These isolates have a high rate of spontaneous mutation during the therapeutic process which induces resistance to Clindamycin. Thus, the empirical treatment options against S. aureus infections have become more limited. Few studies have been performed that report the presence of both constitutive and inducible Clindamycin resistance in India. Therefore, this study was undertaken to detect and report the presence of Clindamycin-resistant phenotypes in a tertiary care hospital, India⁶

 

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Received on 11.06.2020           Modified on 10.08.2020

Accepted on 06.09.2020         © RJPT All right reserved