Title: Analysis of borderline oxacillin resistant Staphylococcus aureus (BORSA) isolated in

JCM Accepts, published online ahead of print on 18 July 2012 J. Clin. Microbiol. doi:10.1128/jcm.01354-12 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Title: Analysis of borderline oxacillin resistant Staphylococcus aureus (BORSA) isolated in Tunisia 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Senda Mezghani Maalej 1, Faouzia Mahjoubi Rhimi 1, Marguerite Fines 2, Basma Mnif 1, Roland Leclercq 2 and Adnene Hammami 1* 1: Laboratoire de Microbiologie, CHU Habib Bourguiba, Sfax, Tunisie 2 : Service de Microbiologie, Université de Caen Basse-Normandie, CHU Cote de Nacre, 14033 Caen cedex, France Running title: Borderline oxacillin resistant Staphylococcus aureus Keywords: Staphylococcus aureus, oxacillin, BORSA, MIC Corresponding author: Pr Adnene Hammami Adresse : Laboratoire de Microbiologie, CHU Habib Bourguiba, Rue El Ferdaous 3029 Sfax, Tunisia. Téléphone : 216 74 456 450 18 Fax : 216 74 456 450 19 E-mail : adnene.hammami@rns.tn 1

20 Abstract 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Twenty three strains of Staphylococcus aureus with borderline resistance to oxacillin were studied. These strains were undetected by the cefoxitin test; PBP2a, meca and meca LGA251 were negative and were genetically unrelated. To detect all of the strains resistant to oxacillin, laboratories should test in routine both cefoxitin and oxacillin. 36 37 2

38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Resistance to methicillin in Staphylococcus aureus is commonly mediated by PBP2a, an additional low affinity penicillin-binding protein, encoded by the chromosomal meca gene (4). In 2011, Garcia-Alvarez et al describe a novel meca gene homologue, called meca LGA251, associated with resistance to ß-lactam antibiotics. This gene has been presented in clinical strains of methicillin-resistant S. aureus (MRSA) which have been isolated in the UK and in Denmark (11). The intrinsic resistance, meca mediated, may be both homogenous which is easily detectable or heterogeneous (4). Oxacillin disc diffusion has been the traditional method for methicillin resistance screening. However, this test often fails to detect heterogeneous MRSA populations. Since 2001, the cefoxitin 30 µg disc test has showed to be more efficient in predicting methicillin resistance (10, 24, 25). This test has been recommended by different committees, such as the CLSI: Clinical and Laboratory Standard Institute (6) and the CA-SFM: Comité de l antibiogramme de la société Française de microbiologie (7), for the purpose of predicting meca-mediated resistance in Staphylococcus spp. Detection of the PBP2a or the meca gene are the reference methods for methicillin susceptibility testing, but these are not feasible in most clinical laboratories (4). Resistance to methicillin may be extrinsic, non meca mediated, in S. aureus with low-level resistance to oxacillin known for borderline oxacillin-resistant Staphylococcus aureus (BORSA) (16-19). Typically, this borderline phenotype results from excess production of ß-lactamase. It was described initially by MC Dougal and Thornsberry in 1986 (17). According to these authors, these strains were neither heteroresistant nor multiple drug resistant, and produced large amounts of normal staphylococcal ß-lactamase which partially hydrolyze oxacillin, and became fully susceptible to oxacillin in the presence of ß-lactamase inhibitors (17). However, the borderline phenotype has been attributed to other mechanisms: production of an 3

61 62 inductible, plasmid-mediated, methicillinase or different modifications in the PBP genes, due to spontaneous amino acid substitutions in the transpeptidase domain (19, 21). 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 In the present report, we analyzed 23 strains of S. aureus with reduced susceptibility to oxacillin that were isolated in Sfax University hospital (Tunisia). From 2006 to 2011, 1895 clinical strains of S. aureus were recovered in Sfax University hospital. Among these strains, 415 (21.9%) were MRSA and 23 (1.2%) had reduced susceptibility to oxacillin. These 23 strains were obtained from various clinical specimens: 14 were from pus, four from Blood culture, three from tracheal aspirate, one from urine and one from ear. Fourteen strains were isolated from the dermatological unit. Antibiotic susceptibility was performed using the disc diffusion method on Mueller-Hinton agar (Bio-Rad). Minimal inhibitory concentrations (MICs) were determined by broth microdilution method in unsupplemented Mueller-Hinton media. Antimicrobial susceptibility results were interpreted according to the CLSI guidelines (6). ß-lactamase production was realized by using nitrocephin discs (cefianse, biomérieux). All of the strains were ß-lactamase producer and were borderline resistant to oxacillin: inhibition zones diameters ranged from 10 mm to 13 mm for oxacillin 1 µg in Mueller-Hinton agar after incubation at 35 C and at 30 C, and MICs varied from 2 to 4 µg/ml. these strains were not only susceptible to cefoxitin (inhibition zone 28 mm) but also susceptible to amoxicillin+clavulanic acid (inhibition zone 25 mm and MICs <0.125-0.25 µg/ml), to cefotaxime (inhibition zone 25 mm and MICs 0.5-2 µg/ml) and to imipenem (inhibition zone 42 mm and MICs <0.125-0.125 µg/ml). 81 82 83 PBP2a production detected using PB2a latex agglutination test as recommended by the manufacturer (biomérieux) and detection of the meca and the meca LGA251 genes by PCR (9, 20) were negative for all of the strains. The lack of the meca gene confirmed that these 4

84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 strains were not MRSA. The phenotype ß-lactamase hyperproducing BORSA was suggested. To confirm this hypothesis, the activity of clavulanic acid in combination with oxacillin was tested as previously demonstrated by others (5,22). We found that these strains became fully susceptible to oxacillin in the presence of ß-lactamase inhibitor. In other words, for all of the strains, a significant increase ( 5 mm) of inhibition zone diameter for oxacillin was showed after the addition of 4 µg of clavulanic acid and a decrease which was more than two fold dilutions in oxacillin MICs was obtained in the presence of 4 µg/ml of clavulanic acid (Table 1). Oxacillin zone diameter and MICs were unaffected by the clavulanic acid in quality control strains (methicillin susceptible S. aureus ATCC 25923 and 29213 and MRSA ATCC 43300) These 23 strains showed different antibiotypes (Table 1). All of these strains were susceptible to fosfomycin, to glycopeptides and to chloramphenicol. The erm ABC genes were investigated for by PCR as described previously (1). ermc was amplified from 13 strains which were resistant to erythromycin. The typing of the strains was performed by pulsed-field gel electrophoresis (PFGE) as described previously (3). Genomic DNA was digested with the restriction enzyme Sma I, and the fragments were separated in agarose gels by electrophoresis according to the manufacturer s recommendations. Image normalization and construction of similarity matrices were carried out using the finger printing II software (Bio-Rad). PFGE revealed that BORSA strains were genetically unrelated in our hospital (Fig 1). 18 genotypically different strains were identified, suggesting that BORSA strains were originated from different ancestors. 5

106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 Borderline strains of S. aureus have been reported to be associated with both nosocomial and community-acquired infections in some institutions, and have been isolated from various infection sites, including skin, surgical wounds, respiratory samples, abscess and blood (2, 12-15, 23). Outbreaks of BORSA infections have been reported in two different dermatological units in Denmark (2, 14). These two outbreaks have been caused by two different clones, based on the same typing methods (PFGE and spa typing). Whereas, in our study, strains that were isolated in the dermatological unit were not closely related. The incidence of BORSA strains is uncommon (19). It s certainly underestimated given that many of clinical microbiology laboratories use only the cefoxitin test for detection of oxacillin resistance in Staphylococcus spp. Indeed, the cefoxitin test is a marker of resistance to oxacillin by acquisition of meca gene and it s unable to detect BORSA strains (10, 24, 25). Detection of BORSA strains may influence the choice of antibiotics in the treatment. It has been augured by some authors that there is no apparent reason for BORSA strains to be considered as resistant to all other ß-lactams (5, 18, 22). Therefore, infections caused by these strains can probably be safely and effectively treated with ß-lactam antibiotics including cloxacillin or with the use of a ß-lactam -ß-lactamase inhibitor combination (16, 26, 27). However, other authors have demonstrated that oxacillin hasn t been effective against BORSA strains (8, 23). As the treatment of infections caused by BORSA with ß-lactams is full of risk, therapy will be conducted according to the MIC of oxacillin and to the severity of the infection. In our study, most of patients were effectively treated with pristinamycin. The five patients who were treated with ß-lactams (cefotaxime or imipenem) were also recovered. 127 128 In conclusion, the clinical laboratories should be in position to recognize BORSA strains by routine susceptibility testing and especially for differentiating them from truly methicillin 6

129 130 131 132 resistant or susceptible S. aureus. Hence, we recommend the utilization of both the cefoxitin disc as a marker of resistance to oxacillin by acquisition of the meca gene, and oxacillin disc on Mueller Hinton agar incubated at 30 C or on Mueller Hinton agar with NaCl for detection of BORSA strains. 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 7

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Dice (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%] pfge pfge PFGE Similarity (%) Strain Pulsotype 50 60 70 80 90 100 FIG. 1. Dendrogram of PFGE pattern of 23 BORSA Strains V20/10 V206/10 UB101/09 N P M UB86/09 K V92/09 L UB15/09 H C4587/08 H V288/06 A V451/06 A V168/07 D V41/07 C T306/07 E N721/11 R V18/11 Q V117/08 F L170/08 F R156/07 B V332/08 G V365/08 G V277/08 G PL1480/08 I V136/10 O V12/09 J

TABLE 1. Characteristics of Borderline oxacillin-resistant S. aureus (BORSA) strains Strain Specimen Date of Inhibition zone diameter (mm) MIC (µg/ml) Ward isolation Other resistance markers a erm PFGE b OXA OXA+CLA FOX AMC CTX IPM OXA OXA+CLA AMC CTX IPM V288 Eye 01/07/2006 Dermatology 10 17 32 29 28 52 4 0.25 0.125 1 <0.125 KAN,TOB,GEN,ERY,L,PT, TET,OFX,RI,FUC ermc A V451 Pus 03/10/2006 Dermatology 11 20 32 30 30 44 4 0.25 0.125 1 <0.125 KAN,TOB,GEN,ERY L,PT, TET,OFX,RIF,FUC R156 Tracheal aspirate 10/01/2007 Intensive care unit 12 20 32 28 27 46 4 0.25 <0.125 0.5 0.125 ERY,TET ermc B V41 Pus 23/01/2007 Dermatology 13 22 32 26 30 48 2 0.125 <0.125 1 <0.125 ERY,TET ermc C V168 Burn 12/04/2007 Dermatology 12 20 30 28 31 48 2 0.25 0.125 1 <0.125 ERY,TET ermc D T306 Pus 23/06/2007 Thoracic surgery 11 17 32 28 26 50 2 0.25 0.125 1 <0.125 TET Neg E V117 Pus 28/03/2008 Dermatology 13 20 32 28 27 48 2 0.25 0.25 0.5 <0.125 KAN Neg F V277 Blood 09/07/2008 Dermatology 12 22 33 30 28 48 2 0.25 0.25 1 <0.125 ERY,L,TET,OFX,RIF,FUC ermc G L170 Ear 21/08/2008 Maxillofacial 12 21 30 30 30 48 4 0.25 0.25 2 <0.125 KAN,TET Neg F V332 Pus 05/09/2008 Dermatology 12 22 30 36 34 52 2 0.125 0.125 1 <0.125 ERY L,TET,RIF,FUC ermc G V365 Pus 17/10/2008 Dermatology 12 21 32 32 30 48 4 0.125 <0.125 1 <0.125 ERY,L,TET,OFX,RIF,FUC ermc G C4587 Pus 01/12/2008 Infectious disease 11 19 31 29 29 46 4 0.125 <0.125 1 <0.125 KAN, L,TET Neg H PL1480 Urine 19/12/2008 Internal Medicine 12 20 30 28 29 46 4 0.5 0.125 2 <0.125 KAN,TET Neg I V12 Pus 10/01/2009 Dermatology 12 23 30 40 38 56 4 0.25 0.125 2 0.125 ERY,L,TET,OFX,RIF,FUC ermc J UB15 Blood 25/01/2009 Burn unit 12 21 28 29 30 49 4 0.125 0.125 1 0.125 KAN,TET Neg H UB86 Tracheal aspirate 13/04/2009 Burn unit 13 21 30 28 30 46 2 0.125 0.125 1 0.125 KAN, ERY,TET ermc K V92 Burn 27/04/2009 Dermatology 12 21 30 32 34 50 4 0.125 0.125 1 0.125 ERY,L,TET,OFX, RIF, FUC ermc L UB101 Tracheal aspirate 27/05/2009 Burn unit 11 20 30 26 32 46 4 0.25 0.25 1 <0.125 TET ermc M V20 Pus 04/11/2009 Dermatology 11 20 30 26 25 46 2 0.25 0.125 1 0.125 TET,OFX Neg N V136 Pus 16/01/2010 Dermatology 13 21 30 30 28 46 2 0.25 0.125 2 <0.125 KAN,TOB,GEN,ERY,L,TET, OFX,RIF,FUC,SXT ermc O V206 Pus 04/05/2010 Dermatology 13 20 30 29 26 48 2 <0.125 0.125 2 <0.125 L,TET,OFX,RIF,FUC Neg P V18 Blood 30/06/2010 Dermatology 12 20 32 25 28 50 2 <0.125 0.125 2 <0.125 KAN,TOB,TET Neg Q N721 Blood 28/01/2011 Neonatology 12 20 30 25 25 50 2 <0.125 0.125 2 <0.125 FUC Neg R ATCC 25923 - - - 22 23 28 36 31 40 - - - - - - - - ATCC 29213 - - - - - - - - - 0.25 0.25 0.25 1 <0.125 - - - ATCC 43300 - - - 12 13 - - - - 16 8 - - - - - - a : OXA: oxacillin, CLA: clavulanic acid, FOX: cefoxitin, CTX: cefotaxime, IPM: imipenem, AMC: amoxicillin-clavulanic acid, KAN: kanamycin, TOB: tobramycin, GEN: gentamicin, ERY: erythromycin, L: lincomycin, PT: pristinamycin, TET: tetracycline, CHL: chloramphenicol, OFX: ofloxacin, RIF: rifampin, FUC: fucidic acid, SXT: trimethoprim-sulfamethoxazole b : PFGE: Pulsed field gel electrophoresis S. aureus ATCC 25923 (MSSA): quality control for disc diffusion; S. aureus ATCC 29213 (MSSA): quality control for MIC; S. aureus ATCC 43300 (MRSA): quality control for oxacillin disc diffusion and MIC 1 ermc A