Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 29  |  Issue : 2  |  Page : 228--233

Inducible clindamycin resistance among clinical isolates of Staphylococcus aureus


Amira A Kilany 
 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shibin Elkom, Menoufia, Egypt

Correspondence Address:
Amira A Kilany
Department pf Clinical Pathology, Faculty of Medicine, Menoufia University, Shibin Elkom, Menoufia, 32511
Egypt

Abstract

Objective: The aim of the study was to detect the presence of inducible clindamycin resistance among clinical isolates of Staphylococcus aureus. Background: The resistance to antimicrobial agents among staphylococci is an increasing problem. This has led to renewed interest in the usage of macrolide–lincosamide–streptogramin B (MLSB) antibiotics to treat S. aureus infections. The resistance to macrolide can be mediated by the msrA gene coding for efflux mechanism or by the erm gene encoding for enzymes that confer inducible or constitutive resistance to MLSB antibiotics. Materials and methods: Ninety staphylococcal isolates were collected and were tested for antibiotic susceptibility using the disk diffusion method. Inducible resistance to clindamycin in S. aureus was tested by using the double disk diffusion test as per CLSI guidelines. Results: Inducible MLSBresistance (EryRClinInd) was seen in 7/90 (7.7%) isolates, the constitutive phenotype of MLSBresistance (EryRClinR) was detected in 6/90 (6.6%) isolates, and the MSBphenotype (EryRClinS) was seen in 34 (37.7%) isolates. In these isolates, inducible clindamycin resistance was detected in 4.3% (3/70) of S. aureus and 20% (4/20) of coagulase-negative staphylococci. Conclusion: This study showed that the double disk diffusion test should be used as a mandatory method in routine disk diffusion testing to detect inducible clindamycin resistance in staphylococci for the optimum treatment of patients.



How to cite this article:
Kilany AA. Inducible clindamycin resistance among clinical isolates of Staphylococcus aureus.Menoufia Med J 2016;29:228-233


How to cite this URL:
Kilany AA. Inducible clindamycin resistance among clinical isolates of Staphylococcus aureus. Menoufia Med J [serial online] 2016 [cited 2020 Sep 20 ];29:228-233
Available from: http://www.mmj.eg.net/text.asp?2016/29/2/228/192418


Full Text

 Introduction



Staphylococcus aureus is a medically important pathogen that is often acquired from hospital settings (nosocomial) as well as from the community (community acquired). Bacteria that reside in anterior nares of hosts serve as reservoirs for the spread of the pathogen and predispose the host to subsequent infections [1].

Antibiotic resistance among the staphylococci has rendered therapy of these infections a therapeutic challenge. Despite early uniform susceptibility to penicillin, staphylococci acquired a gene elaborating b-lactamase that rendered penicillin inactive and that is borne by nearly all clinical isolates. 'Penicillinase-resistant b-lactams' such as methicillin were introduced in the early 1960s, but resistance to them has become an increasing concern. Moreover, once confined to the ecology of hospitals and other institutions, a recent increase in community-acquired methicillin-resistant S. aureus (MRSA) infections has been observed [2].

Empirical treatment options for staphylococcal infections have become limited, as the prevalence of MRSA strains has increased [3].

Macrolides (e.g. erythromycin, clarithromycin), lincosamides, and streptogramin (MLS) antibiotics are structurally unrelated; however, they are related microbiologically because of their similar mode of action. MLS antibiotics inhibit bacterial protein synthesis by binding to 23S rRNA, which is a part of the large ribosomal subunit. They have a spectrum of activity directed against gram-positive cocci [4].

Erythromycin (a macrolide) and clindamycin (a lincosamide) represent two distinct classes of antimicrobial agents that inhibit protein synthesis by binding to the 50S ribosomal subunits of bacterial cells. In staphylococci, resistance to both of these antimicrobial agents can occur through methylation of their ribosomal target site [5].

Different mechanisms of acquired MLS resistance have been found in gram-positive bacteria. The first mechanism of resistance to macrolide described was the post-transcriptional modification of the 23S rRNA by the adenine-N-6-methyltransferase. Target modification alters a site in 23S rRNA common to the binding of MLSB antibiotics. Modification of the ribosomal target confers cross-resistance to MLSB antibiotics (MLSB-resistant phenotype) and remains the most frequent mechanism of resistance. In general, genes encoding these methylases have been designated erm (erythromycin ribosome methylation) [6].

Expression of resistance to MLSB in staphylococci may be constitutive (cMLSB) or inducible (iMLSB). For iMLSB strains, erythromycin will induce the production of methylase, which allows clindamycin resistance to be expressed [7].

 Materials and Methods



Ninety staphylococcal isolates were selected from February 2011 to February 2012. They were recovered from specimens submitted to the Microbiology Laboratory, Menofiya University Hospitals, and were reported as having erythromycin resistance as detected by standard disk diffusion testing according to the method described by Fokas et al. [8].

This study was carried out on 90 patients with clinical isolates of staphylococci; 70 were coagulase-positive staphylococcal isolates (among which 58 isolates were MRSA) and 20 were coagulase-negative staphylococcal isolates. There were 49 male and 41 female patients with ages ranging from 3 to 66 years, giving 64 blood samples, 12 wound samples, and 14 pus samples.

Staphylococcal isolates were identified by routine microbiological tests (Gram stain, catalase and coagulase, and DNase tests).

For all these samples, screening tests were carried out for detection of MRSA as follows:

Culture on ChromID MRSA agar (bioMérieux Marcy l'Etoile, World Headquarters, FRANCE) medium: chromogenic medium was used for the detection of MRSA; Detection of PBP2a (penicillin-binding protein 2a) by Slidex MRSA detection: rapid latex agglutination test was used for the detection of MRSA by detecting PBP2a.

All staphylococcal isolates were tested for antibiotic susceptibility using the double disk diffusion method (D-test) according to CLSI [9]

Mueller–Hinton agar. Antibiotic disks:

[INLINE:1]

Inducible resistance to clindamycin was tested by means of the 'D-test' as per CLSI guidelines. Briefly, the erythromycin (15 μg) disk was placed at a distance of 15 mm (edge-to-edge) from the clindamycin (2 μg) disk on a Mueller–Hinton agar plate, previously inoculated with 0.5 McFarland standard bacterial suspensions. Following overnight incubation at 37°C, flattening of the zone (D-shaped) around clindamycin in the area between the two disks indicated inducible clindamycin resistance, as shown in [Figure 1].{Figure 1}

Three different phenotypes were recognized after testing and then interpreted. This interpretation was done only for erythromycin-resistant S. aureus strains:

MS phenotype: Staphylococcal isolate exhibiting resistance to erythromycin (zone size ≤13 mm) while being sensitive to clindamycin (zone size ≥21 mm) and giving circular zone of inhibition around clindamycin was labeled as having this phenotype. Inducible MLSB (iMLSB) phenotype: Staphylococcal isolate showing resistance to erythromycin (zone size ≤13 mm) while being sensitive to clindamycin (zone size ≥21 mm) and giving D-shaped zone of inhibition around clindamycin with flattening toward the erythromycin disk was labeled as having this phenotype. Constitutive MLSB ( cMLSB) phenotype: This phenotype was labeled for those staphylococcal isolates that showed resistance to both erythromycin (zone size ≤13 mm) and clindamycin (zone size ≤14 mm) with circular shape of zone of inhibition around clindamycin.

Quality control (QC) of the erythromycin and clindamycin disks was performed with S. aureus ATCC25923, according to the standard disk diffusion QC procedure. Additional QC was performed with separate in-house-selected S. aureus strains that demonstrated positive and negative D-test reactions.

 Results



Ninety staphylococcal isolates were tested for susceptibility to erythromycin and other antibiotics by routine disk diffusion testing; 47(52.2%) of them were erythromycin resistant. MLSB-resistant and MSB-resistant phenotypes of s. aureus and S. non-aureus are shown in [Table 1]. The pattern of Ery S Clin S phenotype was detected in 43 (48%) isolates, followed by the MSB phenotype (Ery R Clin S) in 34 (37.7%) and the iMLSB resistance (Ery R Clin Ind) in 7/90 isolates (7.7%), whereas the cMLSB resistance (Ery R Clin R) was seen in 6/90 isolates (6.6%), being the least frequent.{Table 1}

Percentage of both inducible and constitutive resistance was higher among MRSA isolates as compared with methicillin-susceptible Staphylococcus aureus (MSSA), as shown in [Table 2]. The highest percentage of iMLS resistance was in methicillin-resistant coagulase-negative staphylococci (MRCoNS 3) (16%) and MRSA 3 (5.1%), whereas the lowest percentage was in MSSA and methicillin-sensitive coagulase-negative staphylococci (MSCoNS) (0% each). The highest percentage of cMLS was in MRCoNS 4 (21%), followed by MRSA 2 (3.4%), whereas the lowest percentage was in both MSSA and MSCoNS (0% each) and the highest percentage of MSB type was in MRSA 24 (41.3%), followed by MRCoNS 6 (31.5%) and MSSA 4 (33.3%), and the lowest percentage was in MSCoNS (0%).{Table 2}

 Discussion



The discovery and development of antibiotics is undoubtedly one of the greatest advances of modern medicine. Unfortunately, the emergence of antibiotic resistance continues to increase and is acknowledged to be a major threat to the treatment of infectious diseases. Infections caused by antimicrobial-resistant organisms are almost always associated with increased mortality, prolonged hospital stays, and excess costs [0].

Prevalence of the inducible clindamycin (CL)-resistant phenotype varies widely by hospital and geographic region. Failure to identify inducible CL resistance when the ERY R Clin S phenotype is detected may lead to clinical failure of CL therapy [1].

Conversely, labeling all ERY R staphylococci as Clin R or not reporting CL resistance when ERY resistance is present will likely prevent the use of CL in treating infections that would likely respond to CL therapy [8].

Clindamycin is widely used to treat serious staphylococcal infections, particularly in children, because of limited alternative therapy [2]

The aim of the study was to detect the presence of inducible CL resistance among clinical isolates of S. aureus.

This study focused on the existence of staphylococci with inducible clindamycin resistance in Menoufiya University Hospitals. To accomplish this, we performed the D-test as a screening method. To achieve this goal 90 staphylococcal isolates were collected from different specimens such as blood, pus, and sputum of patients who were admitted to different departments in Menoufiya University Hospital from February 2011 to February 2012 on the basis of erythromycin resistance as detected by standard disk diffusion testing. These isolates were identified by morphological, cultural, and biochemical characteristics.

For all these samples screening tests were carried out for the detection of MRSA by culturing on ChromID MRSA agar medium and by detection of PBP2a using Slidex MRSA detection, and isolates were tested for antibiotic susceptibility using the disk diffusion method.

Inducible CL resistance detection was confirmed by using the D-test in which erythromycin 15 mg disk and clindamycin 2 mg disk were placed 15–21 mm edge-to-edge on the Mueller–Hinton agar.

In this study Ery S Clin S was detected in 48% of isolates, followed by the MSB phenotype (Ery R Clin S) in 37.7% and iMLSB resistance (Ery R Clin Ind) in 7.7%, whereas the phenotype of cMLSB resistance (Ery R Clin R) (6.6%) was the least frequent. This result is in agreement with that of Coutinho et al. [3], who found that the MSB-resistant phenotype was more common (11.2%) in relation to the other phenotypes (iMLSB 7.4% and cMLSB 3.2%) in central nervous system isolates studied in Sevilla. These results were to some extent similar to those of Renushri et al. [4], who reported that the iMLSB phenotype was seen in 11.4% of staphylococcal isolates. This similarity in results may be due to the use of the same technique and similar guidelines for drug use.

Various studies have shown the prevalence of the cMLSB phenotype to range from 11 to 27% and the MSB phenotype from 12 to 44% [5].

The results of our study are in contrast to those of Saderi et al. [6], who showed a prevalence of cMLSB, iMLSB, and MSB-resistant phenotypes among erythromycin-resistant S. aureus of 92.9, 6.3, and 0.8%, respectively. Such differences in the MLSB-resistance pattern could be caused by differences in guidelines for drug usage in Iran, where MLSB antibiotics are widely used in treating S. aureus infections.

This study disagrees with the results of the 3-year study by Spiliopoulou et al. [7] on 173 erythromycin-resistant S. aureus strains isolated from patients in a University Hospital in Greece, which reported 61.3, 30.6, and 7.5% prevalence for cMLSB, iMLSB, and MS-resistant phenotypes, respectively. The difference in results may be attributed to the use of a larger number of isolates compared with the present study.

The difference in results may be attributed to various factors – for example, the test procedure factor of interdisk distance; in the present study an interdisk distance of 15 mm was used, which proved to be 100% accurate for the detection of iMLSB. The interdisk distance may give false-negative results if it is more than 15–20 mm. Ajantha et al. [8] found seven iMLSB strains on retesting with 15 mm that were previously reported as D-test negative with 28 mm.

A comparison between CL-resistant phenotypes and methicillin-resistant results in the present study shows that in MRSA the most frequent phenotype was ERY-S, CL-S29 (50%), followed by MSB type 24 (41.3%) and iMLS type 3 (5.1%), whereas the least frequent was the cMLS type 2 (3.4%). In MSSA the most frequent phenotype was ERY-S, CL-S 8 (66.6%), followed by MSB type 4 (33.3%); no other phenotypes were detected. MRCoNS results show that the most frequent phenotypes were ERY-S, CL-S 6 (31.5%) and MSB6 (31.5%), followed by cMLS 4 (21%) and iMLS 3 (16%). In MSCoNS, iMLS 1 (100%) was the only detected type.

These results are in agreement with those described by Merino et al. [9] who found that the rate of iMLSB resistance in erythromycin-resistant strains was significantly higher [7.4%; 5.2% in S. aureus and 14% in coagulase-negative staphylococci (CoNS)] than the rate of cMLSB resistance (3.2%; 1.7% in S. aureus and 7.4% in CoNS), whereas the MSB phenotype was the most common (11.2%; 7.2% in S. aureus, and 23% in CoNS).

Azap et al. [0] also found a relation between iMLS resistance and MRSA in total staphylococcal isolates tested for inducible CL resistance. iMLS resistance was detected in 5.7% of MRSA isolates, in 3.6% of MSSA isolates, in 30.8% of methicillin-resistant CoNS isolates, and in 11.2% of methicillin-sensitive CoNS isolates. These results are in agreement with those of Schreckenberger et al. [1], who found that the iMLSB phenotype was found in 7 and 12% of MRSA and MSSA, respectively, and in 14 and 35% of methicillin-resistant CoNS and methicillin-sensitive CoNS, respectively.

These findings are quite similar to the results of Delialioglu et al. [2] who found that in Turkey 24.3% of S. aureus and 40.2% of CoNS had the phenotype cMLSB, whereas 7.8% of S. aureus and 14.7% of CoNS had an iMLSB phenotype and none of the strains of S. aureus and 18.2% of CoNS strains showed the phenotype MSB. The cMLSB-resistance rate was found to be higher than the rate of inducible resistance. In the MSSA group no constitutive resistance was reported, and inducible resistance to clindamycin was ∼11%.

In contrast, Hamilton-Miller and saroj [3] found that the most common phenotype was iMLSB, whereas the phenotype MSB was present only in 1% of S. aureus and 13% of the CoNS isolates studied, unlike the results of our study, in which the MSB phenotype was more frequent. Moreover, in the present study, the phenotype MSB was found more frequently than the iMLSB phenotype in S. aureus but less frequently in CoNS.

The difference in the results may be attributed to the large number of isolates used in their study compared with the present study as well as to the different techniques employed, as isolates that were found to be clindamycin sensitive or inducible resistant were tested by real-time PCR for erm A gene.

Also, the prevalence rates reported by Yilmaz et al. [4] were higher than those of the present study as their study showed that the inducible Clin R phenotype level was 24.4% among MRSA isolates, 14.8% among MSSA isolates, 25.7% among MRCoNS isolates, and 19.9% among MSCoNS isolates, such differences being caused by differences in guidelines for drug usage in other studies in which MLSB antibiotics were widely used in the treatment of staphylococcal infections.

 Conclusion



Clindamycin is kept as a reserve drug and is usually advocated in severe MRSA infections depending upon the antimicrobial susceptibility results. This study showed that the D-test should be used as a mandatory method in routine disk diffusion testing to detect inducible CL resistance in staphylococci for the optimum treatment of patients.

Recommendations

Clinical microbiology laboratories should use the D-test as standard practice for all erythromycin-resistant staphylococci.Disk placement at a distance of 15 mm provides a more obvious indication of a positive D-zone but requires hand placement of the disks.Real-time PCR should be used for erm A gene detection as it shows a great correlation with D-test results.

Conflicts of interest

There are no conflicts of interest.[24]

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