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Research Article
 

Incidence of Macrolide-lincosamide-streptogramin-B Resistance Phenotypes of Methicillin Resistance Staphylococcus aureus and Methicillin Sensitive Staphylococcus aureus Among Animals in Saudi Arabia



Mohammad Alzohairy
 
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ABSTRACT

Macrolide-lincosamide-streptogramin-B (MLSB) are important antibiotic family for treatment of staphylococcal infections in both humans and animals. Clindamycin is widely used in veterinary medicine to treat a variety of bacterial infections. Thus, this study was conducted to investigate the incidence of MLSB resistance phenotypes among the Staphylococcus aureus isolated from animals by agar disk diffusion (D-test) method. A total of 158 coagulase positive staphylococci were isolated from different stock animals. The incidence rate of MLSB resistance phenotype was 57 (36.1%) in Methicillin-resistant and Methicillin-sensitive Staphylococcus aureus (MRSA and MSSA). Thirty two (20.2%) isolates showed constitutive resistance (cMLSB phenotype) and 12 (7.6%) isolates showed inducible clindamycin resistance (iMLSB phenotype) while the remaining thirteen (8.2%) isolates showed MS phenotype. The rate of constitutive cMLSB phenotypes resistance (32.2%) and inducible clindamycin iMLSB (10%) resistance phenotypes was high in MRSA when compare to MSSA isolates. The higher incidence of inducible clindamycin iMLSB resistance was observed in camel isolates while the lower rate was observed in sheep's isolates. Detection of erythromycin-induced clindamycin resistance in S. aureus by routine screening is necessary so that it will help in guiding therapy and therapeutic failures may be avoided.

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  How to cite this article:

Mohammad Alzohairy , 2012. Incidence of Macrolide-lincosamide-streptogramin-B Resistance Phenotypes of Methicillin Resistance Staphylococcus aureus and Methicillin Sensitive Staphylococcus aureus Among Animals in Saudi Arabia. Research Journal of Microbiology, 7: 256-262.

DOI: 10.3923/jm.2012.256.262

URL: https://scialert.net/abstract/?doi=jm.2012.256.262
 
Received: April 02, 2012; Accepted: July 04, 2012; Published: August 31, 2012



INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) strains have been associated with infections and colonization in human and many animal species (Lowy, 1998; Reacher et al., 2000). Macrolide-lincosamide-streptogramin-B (MLSB) is important antibiotic for treatment of staphylococcal infections in humans and animals. The development of antimicrobial resistance among S. aureus has been associated as important public health concern which causes high morbidity and mortality worldwide. Emergence of methicillin resistance in S. aureus has left us with very few therapeutic alternatives available to treat staphylococcal infections. MLSB family of antibiotics serves as one such alternative. Among this family Clindamycin is a good alternative for the treatment of both methicillin-resistant and -susceptible staphylococcal infections, being the preferred agent due to its excellent pharmacokinetic properties (Frank et al., 2002).

However, the widespread use of MLSB antibiotics has led to an increased number of staphylococcal strains acquiring resistance to MLSB antibiotics. The most common mechanism for such resistance is target site modification mediated by erm genes which can be expressed either as constitutive (constitutive MLSB phenotype) or inducible expression (inducible MLSB phenotype) (Deotale et al., 2010). Strains with inducible resistance to clindamycin are difficult to detect in the routine laboratory as they appear erythromycin resistant and clindamycin sensitive in vitro when not placed adjacent to each other (Fiebelkorn et al., 2003).

There are few epidemiological studies have revealed the occurrence of indistinguishable MRSA clones in animals and in humans exposed to animals, such as veterinarians and farmers (Loeffler et al., 2005; Voss et al., 2005; Weese et al., 2005). Similarly, dogs affected by pyoderma and their owners have been reported to share identical S. intermedius strains in the nasal cavity (Guardabassi et al., 2004). These findings indicate that staphylococci colonizing the nasal mucosa can be transmitted between animals and humans, including clinically relevant bacteria such as MRSA. Clindamycin is a good alternative for the treatment of both methicillin-resistant and -susceptible staphylococcal infections, Veterinarians should be aware of the potential for clinical failure when clindamycin is used to treat staphylococcal infections due to isolates with in vitro inducible clindamycin resistance.

MLSB resistance phenotype frequently is recognized among S. aureus and may be constitutive or inducible. Resistance due to MLSB results from alterations in ribosomal antibiotic binding sites mediated by erythromycin ribosomal methylase (erm) genes. S. aureus with the inducible MLSB phenotype are susceptible to clindamycin and resistant to erythromycin on initial in vitro testing, but may become resistant to clindamycin following exposure to clindamycin. Inducible Clindamycin Resistance (ICR) has been recognized as a cause of treatment failure (Sibery et al., 2003).

Current knowledge on species distribution, diversity and antimicrobial resistance of animal staphylococci is an epidemiological importance. So, the aim of this study was to assess the prevalence and detection of inducible resistance to CL in ER-resistance (ER-R) among animal isolates of S. aureus by D-test.

MATERIALS AND METHODS

Sampling: In the period between January and April, 2010, nasal swabs were collected from camels, sheep, goats and cattle. All nasal swabs were obtained from healthy animals in different areas of Qassim region, Saudi Arabia. For all animals, cotton swabs were rolled on the nasal mucosae of both nostrils and transported in Stuart medium (Oxoid, UK) prior to laboratory analysis as previously described (Alzohairy, 2011).

Bacterial isolates and identification of Staphylococcus aureus: All the samples received in the laboratory were cultured onto various microbiological media. Suspected colonies of Staphylococcus isolates were characterized to species level by Gram stain, catalase, slide and tube coagulase tests (Oxoid, UK) and by biochemical tests (API Staph; bioM´erieux, France).

Identification of MRSA: For the identification of the MRSA among the isolates of S. aureus, ChromID MRSA (Oxoid, Hants, UK) was used. The MRSA only grew on this Hi Chrome MRSA agar, while the MSSA was inhibited on the same agar plate. All cultures showing bright blue colored growth were taken as MRSA positive strains, while all others are recorded as MSSA strains. As controls, all strains were also inoculated on MH Agar and incubated simultaneously.

Additionally, Methicillin-resistance was confirmed via penicillin binding protein 2a (PBP2a) latex agglutination test (PBP20 Test Kit, Oxoid, Hants, UK).

Antimicrobial susceptibility testing: Antimicrobial susceptibility testing was performed by the disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI, 2007) guidelines. The following antimicrobial disks (Oxoid, Hants, UK) were used: chloramphenicol (30 mg), ciprofloxacin (5 mg), clindamycin (2 mg), erythromycin (15 mg), fusidic acid (10 mg), kanamycin (30 mg), mupirocin (5 mg), penicillin (10 U), quinupristin/dalfopristin (15 mg), rifampicin (5 mg), tetracycline (30 mg) and trimethoprim/sulfamethoxazole (1.25+23.75 mg). The reference strain S. aureus ATCC 25923 was used for quality control.

Detection of MLSB phenotypes by double-disc diffusion (D-test) test: Double-disc diffusion testing (D-test) was performed for each isolate according to (CLSI, 2007) guidelines. A 0.5 McFarl and suspension was prepared in normal saline for each isolate and inoculated on Mueller-Hinton agar plate. Clindamycin (CLI)-2 μg and erythromycin (ER)-15 μg disks were placed 15 mm apart edge to edge manually. Following overnight incubation at 37°C, flattening of zone (D shaped) around clindamycin in the area between the two discs, indicated inducible clindamycin resistance. Three different phenotypes were appreciated after testing and the interpretation was done as follows:

Interpretation of the D-test:

Inducible MLSB phenotype: Staphylococcal isolates 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 towards erythromycin disc were labelled as having this phenotype
Constitutive MLSB phenotype: This phenotype was labelled for those Staphylococcal isolates which showed resistance to both erythromycin (zone size = 13 mm) and clindamycin (zone size = 14 mm) with circular shape of zone of inhibition if any around clindamycin
MS phenotype: Staphylococcal isolates exhibiting resistance to erythromycin (zone size = 13 mm) while sensitive to clindamycin (zone size = 21 mm) and giving circular zone of inhibition around clindamycin was labelled as having this phenotype (Fig. 1)

Fig. 1(a-c): Antibiotic susceptibility pattern of isolates, (a) Inducible MLSB phenotype, (b) Constitutive MLSB phenotype and (c) MS phenotype

RESULTS

This study was carried out to determine the incidence of inducible clindamycin resistance staphylococci among animals in Qassim region, Saudi Arabia. A total of 158 S. aureus isolates obtained from consecutive nasal swabs were included, consisting of 90 MRSA and 68 were MSSA (Table 1) . The total rate of MLSB resistance phenotype 57 (36.1%) was observed in both MRSA and MSSA isolates. Among this phenotype 32 (20.2%) had the constitutive resistance phenotype, 12 (7.6%) showed the inducible resistance phenotype and 13 (8.2%) was the MS phenotype (Table 2).

Of the 158 animal isolates of staphylococci studied, 12 (7.6%) showed inducible clindamycin resistance and belonged to the iMLSB phenotypes, Among the iMLSB phenotypes, 9 isolates were MRSA (10%) and 3 were MSSA (4.4%) isolates. Meanwhile, 32 (20.2%) isolates showed constitutive clindamycin resistance and belongs to the constitutive cMLSB phenotype, among them, 29 (32.2%) isolates were MRSA and 3 (4.4%) were MSSA isolates. While remaining 13 (8.2%) showed MS phenotype. Among the MS phenotypes, 9 (10%) isolates were MRSA and 4 (5.8%) were MSSA. As shown in (Table 2).

Among MRSA isolates, the constitutive resistance cMLSB phenotype predominated over the inducible resistance phenotype and susceptible phenotype (32.2, 10 and 10%, respectively). However, the MS phenotype predominated over the inducible resistance iMLSB phenotype and constitutive resistance cMLSB phenotype (5.8, 4.4 and 4.4%, respectively) in MSSA isolates. The Erythromycin-Sensitive and Clindamycin-Sensitive phenotypes was predominated in MSSA isolates (85.29%) when compare to MRSA (47.7%) isolates (Table 2).

The overall resistance pattern for all three phenotypes in isolates according to host animal was as follow; Camel isolates: Total MLSB phenotypes was (53.7%) among these (31.48%) were constitutive resistance cMLSB phenotype, (9.25%) were inducible resistance iMLSB phenotype and (12.96%) were MS phenotype. Sheep isolates: Total MLSB phenotypes (34.21%) that includes (21.05%) constitutive resistance cMLSB phenotype, (5.26%) were inducible resistance iMLSB phenotype and (7.89%) were MS phenotype.

Table 1: Frequency of MRSA and MSSA isolated from animals
MRSA: Methicillin-resistant S. aureus, MSSA: Methicillin-susceptible S. aureus

Table 2: Susceptibility to clindamycin and erythromycin among MRSA and MSSA isolates
MRSA: Methicillin-resistant S. aureus, MSSA: Methicillin-susceptible S. aureus, ER: Erythromycin, CL: Clindamycin, R: Resistant, S: Susceptible

Table 3: Prevalence of clindamycin and erythromycin susceptible phenotypes among staphylococci isolated from studied animals
MRSA: Methicillin-resistant S. aureus, MSSA: Methicillin-susceptible S. aureus, ER: Erythromycin, CL: Clindamycin, R: Resistant, S: Susceptible

Cattle isolates: the total MLSB phenotypes was (27.77%), among them, (13.88%) showed constitutive resistance cMLSB phenotype, (8.33%) were inducible resistance iMLSB phenotype and (5.55%) were MS phenotype. Goat isolates: the total MLSB phenotypes was (16.66%) including (6.66%) constitutive resistance cMLSB phenotype, (6.66%) inducible resistance iMLSB phenotypes and (3.33%) as MS phenotype. Hence, the highest rate of inducible clindamycin resistance iMLSB phenotype prevalence was observed in camel isolates (9.2%) while the lowest inducible clindamycin resistance iMLSB phenotype was observed in sheep isolates (5.3%) (Table 3).

DISCUSSION

The development of resistance in Staphylococcus species to macrolide, lincosamide and streptogramin B has limited the use of these antibiotics (Lewis et al., 2005). Macrolide resistance may be due to enzymes encoded by a variety of erm genes-MLSB phenotype and may be constitutive (cMLSB phenotype) or inducible (iMLSB phenotype). Another mechanism is active efflux pump encoded by the mrs A gene (MS phenotype) (Daurel et al., 2008). Clindamycin is used widely in veterinary medicine to treat a variety of bacterial infections including skin, wound and bone infections, pneumonia, oral cavity infections and infections due to anaerobic bacteria. Clindamycin is a good alternative for the treatment of both methicillin-resistant and -susceptible staphylococcal infections, but therapeutic failures caused by inducible iMLSB phenotype resistance are being reported more commonly (Sibery et al., 2003).

In this study, MRSA isolates had higher constitutive resistance (32.2%) compared to inducible resistance (10%) while in MSSA isolates, constitutive resistance (4.4%) was similar to that of inducible resistance rate (4.4%). These findings go in agreement with several other studies reported before. Azap et al. (2005) found that constitutive phenotype is predominant over inducible phenotype in MRSA isolates and MS phenotypes are only found in MSSA (Kader et al., 2005) also reported (53%) cMLSB and 43% iMLSB (i.e. higher constitutive resistance) among MRSA . Similarly, Gadepalli et al. (2006) showed it to be 30% in MRSA and 10% in MSSA.

On the contrary, several other authors have also reported different results such as Schreckenberger et al. (2004) and Levin et al. (2005) showed higher percentage of inducible resistance in MSSA as compared to MRSA,(7-12% in MRSA and 19-20% in MSSA; 12.5% MRSA and 68% MSSA), respectively. However, taking in account other reports (Rich et al., 2005; Martinez-Aguilar et al. (2003) this study reveals that the rate of inducible resistance varies which highlight the variations and importance of inducible clindamycin resistance in different geographical setting.

There have been a number of reports on the clinical failure due to antibiotics resistant. Rao (2000) and Dinkovic et al. (2001) reported clinical failures after using clindamycin against S. aureus strain which had been demonstrated to express inducible clindamycin resistance phenotype. Additionally, Sibery et al. (2003). also reports another case of clinical failure in which clindamycin resistance developed whilst on therapy for an original clindamycin-susceptible isolate. Therefore veterinarians should be aware of the potential for clinical failure when clindamycin is used to treat staphylococcal infections if the isolates show inducible clindamycin resistance in vitro by D-test.

CONCLUSION

This study reported the incidence of inducible clindamycin resistance in S. aureus isolated from animals in Qassim region of Saudi Arabia. However this study conclude that the routine screening test for detection of erythromycin-induced clindamycin resistance in S. aureus performing by D-test is necessary, so that it will help in guiding therapy and therapeutic failures may be avoided.

ACKNOWLEDGMENTS

Author would like to thank Dr. Habeeb Khadri for the significant input and fertile discussion. This research was supported by Grant Number SR-D-011-626 from the Scientific Research Deanship at Qassim University, Saudi Arabia 2011.

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