O D S b T P A M a b B c d B a A R A A K S S M m B M h 1 B braz j infect dis 2 0 1 8;2 2(2):99–105 www.elsev ier .com/ locate /b j id The Brazilian Journal of INFECTIOUS DISEASES riginal article etection of the mecA gene and identification of taphylococcus directly from blood culture bottles y multiplex polymerase chain reaction aisa Trevizani Rocchetti a,b,∗, Katheryne Benini Martinsa, atricia Yoshida Faccioli Martinsa, Rogério Antonio de Oliveirac, lessandro Lia Mondelli d, Carlos Magno Castelo Branco Fortalezab, aria de Lourdes Ribeiro de Souza da Cunhaa UNESP - Univ Estadual Paulista, Instituto de Biociências de Botucatu, Departamento de Microbiologia e Imunologia, Botucatu, SP, Brazil UNESP - Univ Estadual Paulista, Faculdade de Medicina de Botucatu, Hospital Universitário, Departamento de Doenças Tropicais, otucatu, SP, Brazil UNESP - Univ Estadual Paulista, Instituto de Biociências de Botucatu, Departamento de Biociência, Botucatu, SP, Brazil UNESP - Univ Estadual Paulista, Faculdade de Medicina de Botucatu, Hospital Universitário, Departamento de Medicina Interna, otucatu, SP, Brazil r t i c l e i n f o rticle history: eceived 3 November 2017 ccepted 18 February 2018 vailable online 13 March 2018 eywords: taphylococcus spp. taphylococcus aureus RSA ecA gene a b s t r a c t Introduction: Staphylococcus spp. – both S. aureus, including methicillin-resistant strains (MRSA) and coagulase negative staphylococci (CoNS) – are relevant agents of healthcare- associated infections. Therefore, the rapid recognition of MRSA and methicillin-resistant CoNS from blood stream infections is critically important for patient management. It is worth noting that inappropriate empiric therapy has been associated with higher in-hospital mortality. Material and methods: In this study we evaluated a multiplex polymerase chain reaction (mul- tiplex PCR) standardized to detect Staphylococcus spp., S. aureus, and mecA gene-encoded oxacillin resistance directly from blood culture bottles. A total of 371 blood cultures with Gram-positive microorganisms confirmed by Gram-stain were analyzed. Results from mul- lood cultures ultiplex PCR tiplex PCR were compared to phenotypic characterization of isolates. Results: Staphylococcus aureus was detected in 85 (23.0%) blood cultures and CoNS in 286 (77.0%). There was 100% agreement between phenotypic and multiplex PCR identification. Forty-three (50.6%) of the 85 S. aureus carried the mecA gene and among the 286 CoNS, 225 (78.7%) were positive for the mecA gene. ∗ Corresponding author. E-mail address: taisa texrocchetti@hotmail.com (T.T. Rocchetti). ttps://doi.org/10.1016/j.bjid.2018.02.006 413-8670/© 2018 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. This is an open access article under the CC Y-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). https://doi.org/10.1016/j.bjid.2018.02.006 http://www.elsevier.com/locate/bjid http://crossmark.crossref.org/dialog/?doi=10.1016/j.bjid.2018.02.006&domain=pdf mailto:taisa_texrocchetti@hotmail.com https://doi.org/10.1016/j.bjid.2018.02.006 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ 100 b r a z j i n f e c t d i s . 2 0 1 8;2 2(2):99–105 Conclusions: The multiplex PCR assay developed here was found to be sensitive, specific, rapid, and showed good agreement with the phenotypic results besides being less expensive. This PCR method could be used in clinical laboratories for rapid identification and initiation of specific and effective treatment, reducing patient mortality and morbidity. Furthermore, this method may reduce misuse of antimicrobial classes that are more expensive and toxic, thus contributing to the selection of antibiotic-resistant Staphylococcus spp. © 2018 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ Introduction Sepsis is the systemic response to infection and is the result of the complex interaction between the host’s immune sys- tem and the infecting microorganism.1 Many bacteria are isolated from bloodstream infections, including Gram-positive and Gram-negative bacteria. According to the US Centers for Disease Control and Prevention (CDC),2 a clear change in the nature of the infectious microorganisms has occurred over the past decade, when Gram-positive cocci have exceeded Gram- negative bacilli as the main etiological agents of sepsis and Staphylococcus spp. have become the most prevalent agents. The genus Staphylococcus comprises 52 species3; of these, 17 can be isolated from human biological samples. The genus can be divided into two main groups: coagulase- positive staphylococci, whose main member is S. aureus, and coagulase-negative staphylococci (CoNS). Staphylococcus aureus possesses a wide variety of virulence factors, including peptidoglycan, teichoic acid, enzymes and toxins, adhesion molecules, and four types of cytotoxins.4 They can cause different types of infection, many of them acquired in the hos- pital environment. About 20% to 30% of the human population is colonized with this bacterium and the nasal mucosa is the main ecological niche.5 A relevant factor that complicates treatment of staphylo- coccal infections is the high resistance rate to �-lactam antibi- otics observed among Staphylococcus spp., which requires large-scale use of expensive or toxic antibiotics. Staphylococ- cus spp. resistance to �-lactam antibiotics is mainly due to two different mechanisms which, however, can interact. The first mechanism consists of the production of �-lactamase, an enzyme that hydrolyzes the antibiotic. The second mech- anism is related to a change in the site of action of �-lactams by producing a new penicillin-binding protein, PBP2a, which has low affinity for antibiotics and is absent in suscepti- ble staphylococci.6 PBP2a is encoded by the mecA gene. This gene is located on a mobile genetic element called SCCmec (staphylococcal cassette chromosome mec), which is widely distributed among staphylococci.7 PBP2a acts as a transpep- tidase that resumes cell wall synthesis functions when other PBPs are inhibited, thus ensuring the integrity of the bacterial 8 cell in the presence of �-lactams. In view of the above considerations, rapid detection of bac- teremia and the subsequent rapid identification of the bacteria involved and their susceptibility to antibiotics are of great licenses/by-nc-nd/4.0/). diagnostic and prognostic importance. Multiplex polymerase chain reaction (PCR) allows to amplify genes of multiple pathogens and more than one specific DNA sequence of each pathogen using multiple sets of primers in a single reaction. This technique permits rapid detection and is effective in identifying pathogenic microorganisms in clinical samples, especially those that require urgent initiation of treatment. The aim of the present study was to evaluate the effi- cacy, accuracy and sensitivity of multiplex PCR in detecting Staphylococcus spp., S. aureus, and mecA gene-encoded oxacillin resistance directly from blood culture bottles. Material and methods Isolates A total of 371 Staphylococcus spp. isolated from positive blood cultures between May 2011 and May 2012 were provided by the Laboratory of Microbiology at the Teaching Hospital of Botu- catu Medical School, UNESP - Univ Estadual Paulista (City of Botucatu, São Paulo State, Brazil). Sample collection The isolates included in the study were obtained on the day following growth detection in a Bactec 9120 system and after confirmation of containing probable Staphylococcus spp. by Gram staining (Gram-positive cocci) and positive catalase tests. The blood culture bottles were disinfected with 70% alco- hol and 3 mL of blood was collected with a 5 mL syringe and 70 × 25-mm needle into two microtubes (1.5 mL per micro- tube). One microtube was used for seeding onto blood agar for phenotypic identification and the other microtube was used for extraction of bacterial DNA for multiplex PCR. Phenotypic identification of S. aureus and coagulase-negative staphylococci Phenotypic identification was performed at the Laboratory of Bacteriology, Department of Microbiology and Immunol- ogy, Botucatu Biosciences Institute, UNESP. The samples were seeded into blood agar and incubated for 24 h at 37 ◦C. After isolation from blood agar, the bacteria were submitted to Gram staining for confirmation of morphological staining http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ . 2 0 1 8;2 2(2):99–105 101 c S S D B i t t 1 t E w i t c t i b t 1 T i m p 1 i s c a t w t t ( t t D A T i p n U I S S F p f E 9 9 7 Table 1 – Primers used for the detection of Staphylococcus spp., S. aureus and the mecA gene by multiplex PCR. Gene Primer Amplicon(bp) S. aureus24 SAU327 – GGA CGA CAT TAG ACG AAT CA SAU1645 – CGG GCA CCT ATT TTC TAT CT 1250 16S rRNA25 16S1 – 5′ CCTATAA- GACTGGGATAACTTCGGG 3′ 16S2 – 3′ CTTTGAGTTTCAAC- CTTGCGGTCG 5′ 791 Coa26 COA1 – 5′ GTA GAT TGG GCA ATT ACATTT TGG AGG 3′ COA2 – 5′ CGC ATCAGC TTT GTT ATC CCA TGT A 3′ 117 mecA27 MRS1 – 5′ AAAATC- GATGGTAAAGGTTGGC 3′ MRS2 – 3′ AGTTCT- GCAGTACCGGATTTGC 5′ 533 b r a z j i n f e c t d i s haracteristics, catalase test for characterization of the genus taphylococcus, and coagulase test for differentiation between . aureus and CoNS. NA extraction directly from blood culture efore DNA extraction, the samples were treated to remove nterfering elements in the blood culture. The sample was cen- rifuged at 850 × g for 2 min.9 The supernatant was transferred o another microtube and again centrifuged at 11,000 × g for min. The supernatant was carefully discarded, leaving only he pellet. Five hundred microliters of lysozyme buffer (1 M DTA, 0.1 M NaCl, 10 mM Tris, pH 8.0, and 5% Triton X-100) as then added to the pellet and the mixture homogenized n a vortex. Next, 800 �L benzyl alcohol (Sigma) was added, he mixture homogenized in a vortex, and the microtube entrifuged at 7000 × g for 5 min.10 Two phases formed in he microtube, the lower phase contained the alcohol with nterfering substances (inhibitors) and the upper phase the acteria. For DNA extraction, 320 �L of the liquid was removed from he upper phase and transferred to another microtube and 0 �L lysozyme was added (10 mg lysozyme in 1 mL 10 mM ris–HCl). The microtube was homogenized in a vortex and ncubated at room temperature for 15 min, homogenizing the icrotube at five-minute intervals. After this period, 10 �L roteinase K was added and the mixture was incubated for 5 min at 56 ◦C, homogenizing the microtube at five-minute ntervals in a vortex. The liquid (330 �L) was transferred to a ilica column provided by the Illustra extraction kit (GE Health- are). This column was placed inside a tube and centrifuged t 11,000 × g for 1 min. Lysis solution (500 �L) was added and he column centrifuged at 11,000 × g for 1 min. Next, 500 �L ashing solution was added and the column was again cen- rifuged at 11,000 × g for 3 min. The tube was discarded and he column was transferred to a microtube. Elution solution 200 �L) pre-warmed to 70 ◦C in water bath was then added to he column. After incubation at room temperature for 1 min, he microtube was centrifuged at 11,000 × g for 1 min and the NA obtained was stored in a freezer at −20 ◦C. ntimicrobial susceptibility testing he isolates were submitted to in vitro susceptibility test- ng by the disk diffusion method on Mueller-Hinton agar lates at the Laboratory of Microbiology, Department of Inter- al Medicine, University Hospital, Botucatu Medical School, NESP, according to the Clinical and Laboratory Standards nstitute criteria.11 tandardization of multiplex PCR for the detection of taphylococcus spp., S. aureus, and mecA gene irst, PCR was standardized using a range of annealing tem- eratures to establish the adequate annealing temperature or all primers (Table 1). Amplification was performed in an ppendorf thermocycler. The amplification conditions were: 2 ◦C for 3 min, followed by 30 cycles of DNA denaturation at 2 ◦C for 1 min, annealing at 56 ◦C for 1 min, and extension at 2 ◦C for 3 min. The final reaction volume was 35 �L containing 10.2 �L autoclaved Milli-Q water, 3.5 �L deoxyribonucleotide triphosphates (dNTP) (Life Technologies), 0.8 �L Taq DNA poly- merase (Biotools), 2.5 �L MgCl2-free buffer (Biotools), 2.5 �L MgCl2 (Biotools), 0.14 �M of each 16S primer, 0.86 �M of each MRS primer, 0.35 �M of each SAU primer, 0.57 �M of each COA primer, and 3.5 �L of bacterial DNA. The amplification prod- ucts were analyzed by electrophoresis on 3% agarose gel at 70 V. The following controls were included in all amplification reactions: ATCC 33591 (mecA-positive S. aureus), ATCC 25923 (mecA-negative S. aureus), ATCC 12228 (mecA-negative S. epi- dermidis), and negative control (water). Sensitivity test The sensitivity of multiplex PCR was evaluated by amplifying serial dilutions of S. aureus and CoNS control strains, start- ing with a dilution of 10−1 (corresponding to 107 CFU/mL) until 10−7 (10 CFU/mL) based on a 0.5 McFarland standard (1.5 × 108 CFU/mL). Specificity test The specificity of multiplex PCR was evaluated by PCR using strains of other microbial species isolated directly from a culture and from positive blood culture bottles containing other bacterial species. The following species were analyzed: Enterococcus faecalis, Enterococcus faecium, Streptococcus viridans, Streptococcus pneumoniae, Candida spp., Gram-positive bacilli, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Enterobacter aero- genes, Salmonella spp., Morganella morganii, and Proteus mirabilis. i s . 2 0 1 8;2 2(2):99–105 1 2 3 4 5 6 7 8 9 10 1000 pb 800 pb 500 pb 100 pb Fig. 1 – Electrophoresis gel (3% agarose) of the products of multiplex amplification in blood cultures for the detection of Staphylococcus spp. and the mecA gene. Lane 1: 100–2000 bp Ladder; lane 2: ATCC 33591 (mecA-positive S. aureus); lane 3: ATCC 25923 (mecA-negative S. aureus); lane 4: ATCC 12228 (mecA-negative S. epidermidis); lane 5: negative control; lanes 6 and 7: mecA-positive S. aureus isolate; lanes 8 and 9: mecA-negative CoNS isolate; lane 10: Discussion 102 b r a z j i n f e c t d Detection of ˇ-lactamase hyperproduction and resistance profile confirmation The isolates not carrying mecA gene although resistant to oxacillin by the disk diffusion test were submitted to the fowling tests to confirm if they were �-lactamase hyper- producers, and to confirm the resistance profile: nitrocefin disk test for �-lactamase hyperproduction; simplex PCR for mecA gene detection, cefoxitin by disk diffusion, and amoxicillin/clavulanic acid disk test for resistance confirma- tion. Statistical analysis The kappa statistic was calculated to assess agreement between the methods used for the identification of Staphy- lococcus sp. A level of significance of 5% was adopted for all tests. Statistical analyses were performed using SSPS software version 19.0 (SSPS Inc., Chicago, IL). Results Phenotypic identification and susceptibility tests Identification by the phenotypic method detected 85 (23.0%) S. aureus strains and 286 (77.0%) CoNS strains in the 371 blood cultures analyzed. The disk diffusion test revealed oxacillin resistance in 43 (50.6%) S. aureus isolates and in 236 (82.5%) CoNS isolates. Multiplex PCR Multiplex PCR was standardized for genotypic identification of the genus Staphylococcus (16S rRNA gene), to differentiate coagulase-positive staphylococci and CoNS (coa gene), to iden- tify S. aureus (S. aureus-specific gene), and to detect oxacillin resistance (mecA gene) directly from blood culture bottles (Fig. 1). Amplification was performed on all 371 isolates included in the study. In the case of the 85 S. aureus isolates iden- tified phenotypically, there was 100% agreement with the multiplex PCR results, with amplification of the 16S rRNA, S. aureus and coa genes. Among the 286 CoNS isolates included, 100% agreement between the two methods was only observed in terms of amplification of the 16S rRNA gene. Amplification of the mecA gene was observed in 43 (50.6%) of the 85 S. aureus isolates; of these, 42 were resistant to oxacillin by the disk diffusion test and one isolate was sus- ceptible. Among the 42 (49.4%) mecA gene-negative isolates, 41 were susceptible by the disk diffusion method and one isolate was resistant. The mecA gene was amplified in 225 (78.7%) of the 286 CoNS isolates; of these, 218 were resistant to oxacillin by the disk diffusion test and seven were susceptible. Among the 61 (21.3%) mecA gene-negative CoNS isolates, 43 were clas- sified as susceptible by the disk diffusion test and 18 were resistant. mecA-negative S. aureus isolate. Detection of ˇ-lactamase hyperproduction and resistance profile confirmation Among the 19 isolates not carrying mecA gene although resistant to oxacillin by disk diffusion test, all were posi- tive by nitrocefin disk test and cefoxitin disk diffusion test. On the other hand, six isolates were resistant to amoxi- cillin/clavulanic acid by disk diffusion test. Sensitivity and specificity of the multiplex PCR In the sensitivity test, amplification of fragments was observed up to a dilution of 10−5 (103 CFU/mL) for CoNS and of 10−2 (106 CFU/mL) for S. aureus, as shown in Fig. 2. Analysis of speci- ficity using isolates of other species revealed no amplification of any fragments of the genes investigated by multiplex PCR (Fig. 3). Costs analysis The cost of multiplex PCR and the automated Vitek 2 sys- tem, the system used for direct bacterial identification in blood cultures at the Laboratory of Microbiology, Department of Internal Medicine, University Hospital, Botucatu Medi- cal School, UNESP, was lower than the phenotypic method (Table 2). Among the 371 Staphylococcus spp. positive blood culture sam- ples included in this study, 23% were positive for S. aureus b r a z j i n f e c t d i s . 2 0 1 8;2 2(2):99–105 103 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 CoNS S. aureus Fig. 2 – Sensitivity of multiplex PCR. Electrophoresis gel showing the amplification of different dilutions of CoNS and S. aureus. Lane 1: 100–2000 bp molecular weight marker; 2: mecA-positive S. aureus ATCC; 3: S. epidermidis ATCC; 4: CoNS 10−7 dilution (10 CFU/mL); 5: 10−6 (102 CFU/mL); 6: 10−5 (103 CFU/mL); 7: 10−4 (104 CFU/mL); 8: 10−3 (105 CFU/mL); 9: 10−2 (106 CFU/mL); 10: 10−1 (107 CFU/mL), 11: S. aureus 10−7 dilution (10 CFU/mL); 12: 10−6 (102 CFU/mL); 13: 10−5 (103 CFU/mL); 14: 10−4 (104 CFU/mL); 15: 10−3 (105 CFU/mL); 16: 10−2 (106 CFU/mL); 17: 10−1 (107 CFU/mL); 18: 100–2000 bp molecular weight marker. 1 2 3 4 5 6 7 8 9 1 0 11 12 3 14 15 16 17 18 1 9 20 Fig. 3 – Electrophoresis gel illustrating the specificity of multiplex PCR. Lane 1: 100–2000 bp Ladder; 2: mecA-positive S. aureus ATCC; 3: mecA-negative S. aureus ATCC; 4: CoNS ATCC; 5: Salmonella spp.; 6: Proteus mirabilis; 7: Morganella morganii; 8: Klebsiella pneumoniae; 9: Pseudomonas aeruginosa; 10: Acinetobacter baumannii; 11: Enterobacter cloacae; 12: Enterobacter aerogenes; 13: E. coli; 14: E. faecalis; 15: E. faecium; 16: Streptococcus viridans; 17: Streptococcus pneumoniae; 18: Candida spp.; 19: Gram-positive bacilli. Table 2 – Comparison of the identification costs with the Vitek 2 system and multiplex PCR. Method Cost Vitek identification Total: US$ 6.76 per isolate Blood agar: US$ 0.5 MacConkey: US$ 0.21 Vitek: US$ 6.05 Multiplex PCR identification Total: US$ 4.35 per isolate Extraction: US$ 2.85 Multiplex PCR: US$ 1.21 a l t f 4 t o t r Electrophoresis gel: US$ 0.29 nd 77% were positive for CoNS; of these, 50.6% S. aureus iso- ates and 82.5% CoNS isolates were resistant to oxacillin by he disk diffusion test. In studies conducted with patients rom the hospital where the current study was performed,12 5.1% S. aureus isolates and 72.5% CoNS isolates were resis- ant to this drug. Other studies also reported high rates of xacillin-resistant Staphylococcus spp. isolates.13,14 In view of his increase in resistant isolates, treatment options have nar- owed down to glycopeptides and new synthetic drugs such as linezolid.15 In addition to being expensive and toxic, these drugs select resistant strains. In some countries there are reports of Staphylococcus spp. resistant to vancomycin, which increasingly limits the therapeutic options.16,17 In view of these considerations, the faster the results of culture and antimicrobial susceptibility testing are released, the faster the physician can adjust treatment, reduc- ing the risks and harm caused by the organism to the patient. In the present study, a multiplex PCR assay that permitted direct detection of oxacillin-resistant S. aureus (MRSA) in blood cultures was developed using an extrac- tion technique that was effective and able to eliminate interfering elements present in blood. Bloodstream infec- tions caused by MRSA complicate the prognosis of the patient, increasing the length-of-hospital stay, mortality rate, number of invasive procedures, and hospitalization costs.18 Investigation of the coa gene included in the multiplex assay for detection of all coagulase-positive staphylococci and the use of the S. aureus-specific SAU primer permit to establish whether the isolate is S. aureus or not. i s . 2 r 1 1 1 104 b r a z j i n f e c t d In addition to the detection of Staphylococcus spp., the mul- tiplex assay proposed in this study also included investigation of the mecA gene to detect oxacillin resistance, in order to guide patient treatment at the time of identification. Pereira et al.19 developed a multiplex PCR assay that directly identi- fied three Staphylococcus spp. species, S. aureus, S. epidermidis and S. haemolyticus, and the mecA gene in blood cultures, but this identification was restricted to these species. The mul- tiplex assay standardized in this study permits the detection of all staphylococcal species isolated from bacteremias. The multiplex PCR was tested using 371 samples, 13 blood culture samples containing other bacterial species and bacterial DNA of other species directly from bacterial culture, showed good sensitivity and specificity. In addition to being specific, our multiplex PCR assay exhib- ited a detection threshold of MRSA of 106 CFU/mL. Similar thresholds have been reported in other studies.20 The thresh- old may decrease with increasing number of amplified genes, but even so the threshold found in this study was within the proposed range of detection since, according to Pereira et al.,20 positive blood cultures present a growth of about 109 CFU/mL. The only discrepancy observed in this study was the detec- tion of the mecA gene by the standardized multiplex PCR assay and susceptibility to oxacillin in the phenotypic test, with the mecA gene not being detected in 19 isolates resistant to oxacillin (one S. aureus isolate and 18 CoNS isolates). In these isolates, the mecA gene was detected by simplex PCR, con- firming the results of multiplex PCR. This discrepancy has also been reported in other studies20–23 and may be related to other types of resistance such as �-lactamase hyperproduction or modification of another PBP. Furthermore, eight isolates were positive for the mecA gene and were susceptible in the phe- notypic test (one S. aureus isolate and 7 CoNS isolates). These findings highlight the importance of genotypic techniques for the detection of oxacillin resistance, but do not rule out the need for other phenotypic techniques to initiate more specific and effective treatment. The implementation of a molecular biology technique such as multiplex PCR to assist in the rapid diagnosis of blood- stream infections may be associated with high costs for the institution. A cost analysis performed in the present study showed that the identification of species in culture, i.e., after detection of a positive blood culture with the Bactec system, its seeding on culture plates and identification, which is done with the Vitek 2 system at the Laboratory of Microbiology in Botucatu Medical School has an average cost of US$ 6.76. In the case of multiplex PCR, which does not require seeding of the blood culture for isolation of the microorganisms since it uses direct identification from blood culture bottles, DNA extrac- tion and PCR amplification for detecting Staphylococcus spp., S. aureus, and the mecA gene were estimated to cost US$ 4.35 per isolate. The multiplex PCR assay developed here was found to be sensitive, specific, rapid and less expensive than the pheno- typic technique used, and showed good agreement with the phenotypic results. This method could be used in clinical lab- oratories for rapid identification and initiation of specific and effective treatment, reducing patient mortality and morbid- ity. Furthermore, this method may reduce the misuse of other antimicrobial classes that are more expensive and toxic, thus 1 0 1 8;2 2(2):99–105 contributing to the selection of antibiotic-resistant Staphylo- coccus spp. Conflicts of interest The authors declare no conflicts of interest. Acknowledgements São Paulo Research Foundation (FAPESP Grant 2010/14250-0) and National Council for Scientific and Technological Devel- opment (CNPq) for financial support. e f e r e n c e s 1. Russel JA. Management of sepsis. N Engl J Med. 2006;355:1699–713. 2. Magill SS, Edwards JR, Bamberg W, et al. Multistate point-prevalence survey of health care-associated infections. N Engl J Med. 2014;370:1198–208. 3. Euzeby J. List of Prokaryotic Names with Standing in Nomenclature: LPSN; 2017. Available from: http://www.bacterio.net/s/staphylococcus.html 4. Bannerman TL. Staphylococcus, Micrococcus and the other catalase-positive cocci that grow aerobically. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, editors. Manual of clinical microbiology. 8th ed. Washington, DC: ASM Press; 2003. 5. Springer B, Orendi U, Much P, et al. Methicillin-resistant Staphylococcus aureus: a new zoonotic agent? Wien Klin Wochenschr. 2009;121:86–90. 6. Hackbarth CJ, Chambers HF. Methicillin-resistant Staphylococci: genetics and mechanisms of resistance. Antimicrob Agents Chemother. 1989;33:991–4. 7. Lim TT, Coombs GW, Grubb WB. Genetic organization of mecA and mecA-regulatory genes in epidemic methicillin-resistance S. aureus from Australia and England. J Antimicrob Chemother. 2002;50:819–24. 8. Kaiser TDL, Pacheco FC, Lima AA, Pereira EM, Santos KRN, Nunes APF. Avaliação de métodos comumente usados em laboratório para a determinação da suscetibilidade à oxacilina entre amostras de Staphylococcus spp., isoladas de um Hospital de Vitória, estado do Espírito Santo. Soc Bras Med Trop. 2010;43:298–303. 9. Hogg GM, Mckenna JP, Ong G. Rapid detection of methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive BacT/Alert ® blood culture bottles using real-time polymerase chain reaction: evaluation and comparison of 4 DNA extraction methods. Diagn Microbiol Infect Dis. 2008;61:446–52. 0. Fredricks DN, Relman DA. 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