Identification and detection of methicillin resistance in Non-Epidermidis coagulase-negative staphylococci

316 BJID 2008; 12 (August) Identification and Detection of Methicillin Resistance in Non-Epidermidis Coagulase-Negative Staphylococci Carina Secchi1,2, Ana Lúcia Souza Antunes1, Leandro Reus Rodrigues Perez 1, Vlademir Vicente Cantarelli2,3 and Pedro Alves d’Azevedo1 1 Post Graduation Program in Medical Sciences of Federal University of Health Sciences of Porto Alegre (UFCSPA); 2Weinmann Laboratory; 3 Feevale Universitary Centre, NH; Porto Alegre, RS, Brazil The NCCLS (2004) presented a new methodology to detect, by disk-diffusion agar, oxacillin-resistance using a cefoxitin disk. We identified coagulase-negative staphylococci (SCoN) to the species level and compared the use of cefoxitin disks (30 µg) with oxacillin disks (1 µg), agar dilution (minimum inhibitory concentration of oxacillin) and mecA gene detection in isolates of coagulase-negative bacteria other than Staphylococcus epidermidis (SCoNne). A total of 238 SCoNne was evaluated; oxacillin-resistance (the mecA gene) was detected in 71% of the isolates. All methods gave 100% sensitivity, based on presence of the mecA gene. The specificity of the cefoxitin disk was 100%, while the oxacillin disk gave a specificity of 91% and agar dilution oxacillin gave a specificity of 88%. We conclude that the cefoxitin disk is an efficient test, and it is an easy method for use in clinical laboratories to detect oxacillinresistance in staphylococci. Key-Words: Coagulase-negative Staphylococcus, cefoxitin, oxacillin, mecA gene, susceptibility diagnostic. Coagulase-negative staphylococci (SCoN) are common pathogens of the blood stream, being frequently related to nosocomial infections, especially in neonates and immunocompromised patients; transmission usually involves medical devices, such as catheters and prostheses [1-3]. Correct identification of SCoN species has become important in clinical laboratories, since several species have been recognized as potential pathogens, especially in a nosocomial setting [4]. Although Staphylococcus epidermidis causes most SCoN infections, many other species have been identified in association with human infections, for example, Staphylococcus lugdunensis, associated with native valve endocarditis and Staphylococcus haemolyticus, which can be multiresistant, including reduced susceptibility to vancomycin [5-7]. Methicillin-resistant staphylococci are considered important agents of nosocomial infections and have frequently been isolated in hospitals throughout the world, including Brazilian hospitals [8]. Sader et al. reported that 80% of SCoN recovered from blood in Latin America were oxacillin resistant [9]. Susceptibility testing by phenotypic methods can be problematic for the detection of methicillin resistance in SCoN because of heterogeneous expression in many strains, affected by growth conditions and by the nature of the beta-lactam agents that are used [10]. For this reason, mecA gene detection by PCR is considered the gold standard for methicillin resistance detection in Staphylococcus spp. [11]. To improve accuracy in the detection of resistance, NCCLS Received on 2 March 2008; revised 20 July 2008. Address for correspondence: Dr. Carina Secchi. Rua Ramiro Barcelos, 910/5ºfloor. Phone: 55-051-33143846. Fax: 55-051-33117813. Porto Alegre, RS, Brazil, Zip code: 90035001.E-mail: . Financial support: CNPq, UFCSPA, Weinmann Laboratório. Part of this paper was presented at the 107th General Meeting of the American Society for Microbiology (Toronto, Can, 2007). The Brazilian Journal of Infectious Diseases 2008;12(4):316-320. © 2008 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved. 2004 recommended that clinical laboratories should use cefoxitin disk (30 µg) tests for detection of oxacillin resistance in Staphylococcus spp. [12]. Several studies have been performed to compare results obtained with cefoxitin and oxacillin disks and how they correlate with the presence of the mecA gene in Staphylococcus spp. [13-15]. We identified all SCoN to the species level and compared the use of cefoxitin disks (30 µg) with oxacillin disks (1 µg), agar dilution (MIC of oxacillin), and mecA gene detection, in isolates of coagulasenegative other than Staphylococcus epidermidis (SCoNne). Material and Methods Bacterial Isolates A total of 238 samples of SCoNne were analyzed, from a collection of samples of SCoN of the Gram-positive Cocci Laboratory of the UFCSPA, stored in skim milk (Difco, Detroit) at –20°C. The samples were obtained from blood cultures collected consecutively, between 2002 and 2004, in the Complexo Hospitalar Santa Casa, Porto Alegre, RS, Brasil. Identification of Isolates The isolates were cultured in Tryptic Soy agar (Oxoid, Basingstoke, UK), supplemented with 5% sheep blood, for 24h at 35°C; colony morphology, hemolytic activity and purity were evaluated. Subsequently, phenotypic tests were evaluated by the conventional method proposed by Kloss & Bannerman 1994, and modified by Bannerman 2003, which consists of a set of biochemical tests that determine the utilization of coagulase, catalase, alkaline phospatase, ornithine decarboxylase, urease, PYR (pyrrolidinyl-βnaphthylamide hydrolysis), and acid production from carbohydrates (trehalose, mannitol, mannose, sucrose, maltose, lactose, and cellobiose) [16,17]. Anaerobic growth in thioglycolate and susceptibility to novobiocin, polymyxin B, bacitracin, desferrioxamine and fosfomycin using disk diffusion tests were evaluated [18]. Quality control was performed with S. epidermidis ATCC 12228. The samples that gave variable results in the phenotypic test www.bjid.com.br BJID 2008; 12 (August) Detection of Methicillin Resistance in SCoNne identifications, or to confirm less frequent species, were run through an automated method of identification (Microscan Walkway; Dade Behringer, Deerfield, IL, USA). The automated results that gave a low percentage certainty of species identification were submitted to determination of the sodA gene by PCR amplification and sequencing with specific primers: d1: 5’CCITAYICITAYGAYYGCIYTIGARCC3’ and d2: 5’ARRTARTAIGCRTGYTCCCAIACRTC-3’ [19]. Disk Diffusion Test (DD) The suspensions were adjusted to a 0.5 McFarland standard for each sample to perform the disk diffusion method (Kirby-Bauer) on Mueller-Hinton agar plates (Difco, Laboratories, Detroit, Mich), using cefoxitin (30 µg) and oxacillin (1 µg) disks (Oxoid, Basingstoke, UK), according to the criteria recommended by CLSI 2005 [20]. The plates were incubated at 35° C and screened after 24h. Agar Dilution Test Determination of the minimum inhibitory concentration (MIC) for oxacillin was performed by bacterial suspension (0.5 McFarland), diluted 1:10 in saline solution and inoculated on Mueller-Hinton agar plates supplemented with 2% NaCl by using Steers replicator. Concentrations of 0.125µg/mL-4 µg/mL of oxacillin (Sigma Chemical Co, St. Louis, USA) were used for determination of the MIC of oxacillin. (...truncated)