Prevalence of Phenotypic Resistance of Staphylococcus Aureus Isolates

l E l TIC

Prevalence of phenotypic resistance of Staphylococcus aureus IGINA r r A isolates to macrolide, lincosamide, streptogramin B, ketolid O and linezolid antibiotics in Turkey

ABSTRACT Authors Riza Adaleti1 The incidence of drug-resistant pathogens differs greatly between countries according to differ- Yasar Nakipoglu2 3 ences in the usage of antibiotics. The purpose of this study was to investigate the phenotypic re- Nurgul Ceran Cihan Tasdemir1 sistance of 321 methicillin resistance Staphylococcus aureus (MRSA) and 195 methicillin suscepti- Fatma Kaya1 ble S. aureus (MSSA) in a total of 516 S. aureus strains to macrolide, lincosamide, streptogramin B Semiha Tasdemir1 (MLS ), ketolid, and linezolid. Disk diffusion method was applied to determine MLS phenotype B B 1Clinical Microbiology and susceptibility to different antibiotic agents. It was found that 54.6% of the isolates were resist- Laboratory, Haydarpasa ant to erythromycin (ERSA), 48% to clindamycin, 55% to azithromycin, 58.7% to spiramycin, Numune Education 34.7% to telithromycin, and 0.4% to quinupristin-dalfopristin, respectively. No strain resistant to and Research Hospital, Istanbul, Turkey. 2 linezolid was found. The prevalence of constitutive (cMLSB), inducible (IMLSB), and macrolides Istanbul University, and type B streptogramins (M/MS ) among ERSA isolates (237 MRSA, 45 MSSA) was 69.6 %, Istanbul Faculty of B Medicine, Department of 18.2%, and 12.2 % in MRSA and 28.9%, 40%, and 31.1% in MSSA, respectively. In conclusions, Microbiology and Clinical the prevalence of cMLS was predominant in MRSA; while in MSSA strains, iMLS and M/MS Microbiology, 34390 Capa, B B B Istanbul, Turkey. phenotype were more higher than cMLSB phenotype resistance. The resistance to quinupristin- 3Clinical Microbiology dalfopristin was very low, and linezolid was considered as the most effective antibiotic against all and infectious disease, S.aureus strains. Haydarpasa Numune Education and Research Hospital, Istanbul, Turkey. Keywords: Staphylococcus aureus, macrolide, lincosamide, streptogramin B, ketolid, linezolid, MLSB. [Braz J Infect Dis 2010;14(1):11-14]©Elsevier Editora Ltda.

INTRODUCTION omal mutations and antibiotic inactivation by specific hydrolases or phosphotransferases.3 Ke- Macrolides (e.g., erythromycin, azithromycin, tolides belong to the MLSB family, and telithro- spiramycin), lincosamides (e.g., clindamycin, lin- mycin is the first commercially available ketolide.4 comycin), and streptogramin B (e.g., quinupris- Oxazolidinones and specifically linezolid are tin) are groups of antibiotic collectively named new class of compounds that binds to the 23S MLS .1 They are chemically distinct, but have B portion of the 50S ribosomal subunit, prevent- similar inhibitory effects on bacterial protein syn- ing initiation complex formation with activity Submitted on: 04/13/2009 thesis. MLSB, commonly used in treatment of sta- against methicillin-resistant S. aureus (MRSA) 1 Approved on: 10/13/2009 phylococcal infections, and clindamycin is a fre- and vancomycin-resistant Enterococcus spp. quent choice for some staphylococcal infections, 5 Correspondence to: (VRE). Quinupristin-dalfopristin (Synercid, Dr. Yasar Nakipoglu particularly skin and soft-tissue infections, and it 30:70 ratio) is the first parenteral streptogramin Department of is an alternative in the penicillin-allergic patients.2 that has recently been licensed for clinical use Microbiology and Clinical The macrolide antibiotic resistance in S. aureus is Microbiology in the United States and Europe for the treat- Istanbul Faculty of usually caused either by ribosomal modification ment of infections caused by multidrug resist- Medicine mediated by 23S rRNA methylases encoded by ant and Gram-positive pathogens.6 Istanbul University erm genes, or by active efflux of the antimicrobial 34390 Capa Quinupristin and dalfopristin enter bacterial Istanbul, Turkey. agent by an ATP-dependent pump encoded by cells by diffusion and bind to different sites on the Tel: +90 212 414 20 00- msrA gene. Methylases confer inducible (iMLS ) 50S ribosomal subunit, resulting in an irrevers- 32372, Fax: +90 212 414 B 20 37. E-mail: yasarnakip@ or constitutive (cMLSB) resistance, while the ef- ible inhibition of bacterial protein synthesis. The yahoo.com flux mechanism affects only macrolides and type combination synergistic effect appears to result B streptogramins (M/MS ). Other more rare from the fact that these compounds target early B We declare no conflict 7 macrolide resistance mechanisms include ribos- and late steps in protein synthesis. of interest.

11 Prevalence of MLSB, ketolid, and linezolid antibiotics resistance in Staphylococcus aureus strains

In vitro tests show that strains with constitutive clindamycin, 284 (55%) to azithromycin, 303 (58.7%) to resistance are resistant to all macrolides, which comprise spiramycin, 179 (34.7%) to telithromycin, and two (0.4%) 14-(e.g.erythromycin), 15-(e.g.azithromycin), and 16- strains to quinupristin-dalfopristin. No strain resistant to membered rings (e.g. spiramycin), lincosamides, and strep- linezolid was found. As for phenotypic resistance of ERSA togramin B, while inducibly-resistant strains are resistant isolates, the rate of cMLS , iMLS , and M/MS in 282 ERSA 3 B B only to 14- and 15- membered-ring macrolides. The ob- strains was 178 (63%) cMLS , 61 (22%) iMLS , and 43 (15%) jective of the present study was to investigate prevalence of B B M/MS , respectivley. The distribution of cMLS , iMLS , MLS , ketolid, and linezolid phenotypic resistance in clinical B B B B and M/MS in ERSA-MRSA isolates was 69.6 %, 18.2%, S. aureus strains. B and 12.2 %; and in ERSA-MSSA isolates it was 28.9%, 40%, MATERIAL AND METHODS and 31.1%, respectively, which showed a predominance of

cMLSB in MRSA, while iMLSB and M/MSB phenotypic re- Bacterial strains sistance patterns were higher in MSSA isolates (Table 1). Between January 2006 and April 2007, 321 MRSA and 195 DISCUSSION MSSA, a total of 516 S. aureus isolates were obtained from different clinical specimens at Haydarpasa Numune Education This study was conducted at the largest educational hospi- and Research Hospital in Istanbul, Turkey. The isolates were tal in Istanbul-Turkey to investigate the prevalence of MLSB, identified according to Gram stain, catalase, and coagulase ketolid, and linezolid in 516 S. aureus isolates. The preva- production (Slidex Staph Plus, Biomérieux, France). Dupli- lence of ERSA in Turkish isolates was found to be higher cate isolates was not included. S. aureus ATCC 25923 was used (54.6%) than those obtained (39%) in a study perfomed as quality control in susceptibility testing. by the neighbours of Turkey with the participation of 20 Antimicrobial disks European university hospitals.10 This differance is more Antimicrobial disks were purchased from Oxoid (Hemakim, likely attributed to the high proportion of MRSA (62.2%) Istanbul, Turkey). in our S .aureus isolates compaed to that of the European study (22%). However, they also reported10 higher rate of Determination of antimicrobial susceptibility test cMLSB in MRSA (93%) and MSSA (44%) than we obtained 11 and MLSB phenotype resistance patterns in MRSA (69.6%) or in MSSA isolates (28.9%). Aktas et al.

MLSB phenotype resistance pattern was determined conducted a study at the University hospital in Turkey on according to the method advised by Clinical and Laboratory only 22 MRSA and found that 63%, 18%, and 18% of the 8 Standards Institute (CLSI). Briefly, an overnight culture of isolates exhibited cMLS , iMLS , and M/MS , respectively. 8 B B B each isolate was adjusted to 0.5 McFarland (10 cf/mL) and Spiliopoulou et al.12 have mentioned in a study on ERSA spread on unsupplemented Mueller-Hinton agar (HIME- strains that only 5.3% of MRSA isolates expressed iMLS DIA, Himedia Laboratories, Mumbai , India). The following B and the rest displayed cMLS , while in MSSA, 78.3% were antibiotic disks were applied on an inoculated media: azi- B iMLS and 21.7% were M/MS , similar with our finding in thromycin (Az-15 μg), spiramycin (Sp 100 μg), telithromy- B B which the percentage of M/MS (31.1%) in MSSA was two- cin (Te-15 μg), quinupristin-dalfopristin (Q-D-15 μg), and B linezolid (Li-30 μg), erythromycin (E-15 μg), and clindamy- fold higher (12.2%) than in MRSA. A study of Janapatla et 13 cin (Cl-2 μg) disks were placed by hand to provide distances al. from Taiwan reported that iMLSB was predominant in of 15-26 mm from edge to edge. Following incubation for MSSA (8%) than in MRSA (4%). Otsuka et al.14 have also re- 16 to 18 hours at 35° C, zone diameters were measured in ported that 61.3% of the Japanese MRSA isolates expressed

the usual manner; any flattening or blunting of clindamycin cMLSB and 94% of the MSSA isolates displayed iMLSB. 15 zone shape (D-shape), indicating iMLSB, while resistance to A retrospective study conducted by Modak et al. on 13,946

both erythromycin and clindamycin indicated cMLSB. Lack S. aureus strains collected between 1994-2005 revealed a of a D-shaped zone in erythromycin resistant and clindamy- stable incidence of cMLS strains, but also a significant in- cin-susceptible isolates were interpreted as M/MS . Due to B B crease in the incidence of isolates that were susceptible to lack of CLSI zone diameters criteria for spiramycin, we used clindamycin and resistant to erythromycin, and in iMLS . the Comite´ de l’Antibiogramme de la Socie´te´ Française de B They attributed this high incidence to the increased use of Microbiologie recommendation of zone diameters ≥ 24 mm macrolides and clindamycin during the same period. Me- as susceptible, and < 19 mm as resistance.9 16 rino-Díaz et al. reported that the rate of iMLSB resistance was significantly higher in S. aureus (5.2%) than the rate of RESULTS cMLSB (1.7%) in cutaneous strains from Spain. On the other Of the 516 isolates, 237 MRSA and 45 MSSA, a total of hand, 41.5% of the ESSA isolates (44.7% of MSSA and 35.7% 282 (54.6%) S. aureus isolates were found to be resistant of MRSA) were highly resistant to spiramycin, in contrast to erythromycin (ERSA) and the rest was susceptible to to the low resistance rate to clindamycin (4%) and azithro- erythromycin (ESSA), 248 (48%) isolates were resistant to mycin (1%). The ribosomal mutations and antibiotic inac-

12 Adaleti, Nakipoglu, Ceran et al.

tivation are the mechanisms that might play a role in the last 3 resistance of ESSA and differ from MLS . Only 0.4% of the S. (0)

(%) B (55) (48) (0.4) (34.7) (58.7) (54.6) aureus isolates were resistant to quinupristin-dalfopristin and strains 0 2 n

this agent was effective in all of the examined isolates. There 179 303 284 248 282 Total was concern regarding the use of streptogramin antibiotic 0 0 13 82 45 40 45 (virginiamycin) as a feed additive in the animal husbandry aureus MSSA

(n=195) 17 (n= 516) and development of cross-resistance against this antibiotic. Although the first resistance to linezolid was reported in 2001 Total S. Total 0 2 18

166 221 239 208 237 due to mutations in the 23S rRNA, we did not detect any M r SA (n=321) resistance or even decreased susceptibility to this antibiotic,

and most reports have shown that the resistance rate to this (0) (0) (0) (1) (4) (0) (%) S. aureus S. 18-20

(41.5) agent is still low. Our study and review of the studies re- 0 0 0 2 9 0 n 97 lated to macrolides resistance in S. aureus demonstrated that Total methicillin resistance leads physicians to use different mac- 0 0 0 0 9 0 67

MSSA rolides, mainly eryhromycin, azithromycin, and spiramycin or (n=150) lincosamides, such as clindamycin and lincomycin which facili- (ESSA: n=234) 0 0 0 2 0 0 30 tate development of different MLSB phenotypic patterns, and M r SA (n=84) which mostly end with resistance to macrolides, lincosamide,

streptogramin B, and ketolid (cMLSB). We believe that this is Erythromycin susceptible susceptible Erythromycin (0) (0) (0) (0) (%)

(21) the reason behind the increased prevalence of cMLS in geo- (100) (100) B graphical area with high prevalence of MRSA, and vice versa. 0 0 0 9 0 n 43 43 Our study showed that the prevalence of MLSB in Total Turkish S. aureus isolates was high and that the predominant 0 0 2 0 0 14 14 phenotype was cMLS in MRSA and iMLS and M/MS in MSSA B B B (n=14) MSSA isolates, which is in agreement with reports of most 0 0 0 7 0 29 29 countries. Linezolid and quinupristin-dalfopristin were very M r SA (n=29) effective and promising. The accurate use of these new agents

M/MSB (n=43) might avoid treatment failure especially in macrolid-resistant (0) (0) (2) (%) (31) (100) (100) (100) S. aureus infections. 0 0 1 n 19 61 61 61

(E r SA: n= 282) REFERENCES Total 0 0 0 0 18 18 18 1. Zelazny AM, Ferraro MJ, Glennen A et al. Selection of Strains MSSA (n=18) for Quality Assessment of the Disk Induction Method for S.aureus

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cM l SB (n=178) tion 2005; 11(4):337-40. 0 2 4. Davis KA, Crawford SA, Fiebelkorn KR, Jorgensen JH. Selec- 165 165 165 165 165 M r SA (n=165) tion of Strains for Quality Assessment of the Disk Induction

Method for Detection of Inducible Clindamycin Resistance in Staphylococci: a CLSI Collaborative Study, Antimicrob Agents Chemother 2005; 49(7):3059-61. 5. Swaney SM, Aoki H, Ganoza MC, Shinabarger DL. The oxa- zolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrob Agents Chemother 1998; 42:3251-5. 6. Raad I, Hachem R, Hanna H. Treatment of vancomycin-resistant enterococcal infections in the immunocompromised host: quinupristin-dalfopristin in combination with minocycline. Linezolid Quinupristin-dalfopristin Telithromycin Spiramycin Azithromycin Clindamycin Erythromycin Antibiotic

Antimicrob. Agents Chemother 2001; 45:3202-4. Table 1. Resistance of S. aureus Strains with different MLSB phenotypic patterns to various antibiotics

Braz J Infect Dis 2010;14(1):11-14 13 Prevalence of MLSB, ketolid, and linezolid antibiotics resistance in Staphylococcus aureus strains

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