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Detection of the Meca Gene and Identification of Staphylococcus braz j infect dis 2 0 1 8;2 2(2):99–105 The Brazilian Journal of INFECTIOUS DISEASES www.elsevier.com/locate/bjid Original article Detection of the mecA gene and identification of Staphylococcus directly from blood culture bottles by multiplex polymerase chain reaction a,b,∗ a Taisa Trevizani Rocchetti , Katheryne Benini Martins , a c Patricia Yoshida Faccioli Martins , Rogério Antonio de Oliveira , d b Alessandro Lia Mondelli , Carlos Magno Castelo Branco Fortaleza , a Maria de Lourdes Ribeiro de Souza da Cunha a UNESP - Univ Estadual Paulista, Instituto de Biociências de Botucatu, Departamento de Microbiologia e Imunologia, Botucatu, SP, Brazil b UNESP - Univ Estadual Paulista, Faculdade de Medicina de Botucatu, Hospital Universitário, Departamento de Doenc¸as Tropicais, Botucatu, SP, Brazil c UNESP - Univ Estadual Paulista, Instituto de Biociências de Botucatu, Departamento de Biociência, Botucatu, SP, Brazil d UNESP - Univ Estadual Paulista, Faculdade de Medicina de Botucatu, Hospital Universitário, Departamento de Medicina Interna, Botucatu, SP, Brazil a r t i c l e i n f o a b s t r a c t Article history: Introduction: Staphylococcus spp. – both S. aureus, including methicillin-resistant strains Received 3 November 2017 (MRSA) and coagulase negative staphylococci (CoNS) – are relevant agents of healthcare- Accepted 18 February 2018 associated infections. Therefore, the rapid recognition of MRSA and methicillin-resistant Available online 13 March 2018 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 Keywords: mortality. Material and methods: In this study we evaluated a multiplex polymerase chain reaction (mul- Staphylococcus spp. Staphylococcus aureus tiplex PCR) standardized to detect Staphylococcus spp., S. aureus, and mecA gene-encoded MRSA oxacillin resistance directly from blood culture bottles. A total of 371 blood cultures with mecA gene Gram-positive microorganisms confirmed by Gram-stain were analyzed. Results from mul- Blood cultures tiplex PCR were compared to phenotypic characterization of isolates. Multiplex PCR 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 [email protected] (T.T. Rocchetti). https://doi.org/10.1016/j.bjid.2018.02.006 1413-8670/© 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/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/ licenses/by-nc-nd/4.0/). diagnostic and prognostic importance. Multiplex polymerase Introduction chain reaction (PCR) allows to amplify genes of multiple pathogens and more than one specific DNA sequence of each Sepsis is the systemic response to infection and is the result pathogen using multiple sets of primers in a single reaction. of the complex interaction between the host’s immune sys- This technique permits rapid detection and is effective in 1 tem and the infecting microorganism. Many bacteria are identifying pathogenic microorganisms in clinical samples, isolated from bloodstream infections, including Gram-positive especially those that require urgent initiation of treatment. and Gram-negative bacteria. According to the US Centers for The aim of the present study was to evaluate the effi- 2 Disease Control and Prevention (CDC), a clear change in the cacy, accuracy and sensitivity of multiplex PCR in detecting nature of the infectious microorganisms has occurred over the Staphylococcus spp., S. aureus, and mecA gene-encoded oxacillin past decade, when Gram-positive cocci have exceeded Gram- resistance directly from blood culture bottles. negative bacilli as the main etiological agents of sepsis and Staphylococcus spp. have become the most prevalent agents. 3 Material and methods The genus Staphylococcus comprises 52 species ; of these, 17 can be isolated from human biological samples. The Isolates genus can be divided into two main groups: coagulase- positive staphylococci, whose main member is S. aureus, A total of 371 Staphylococcus spp. isolated from positive blood and coagulase-negative staphylococci (CoNS). Staphylococcus cultures between May 2011 and May 2012 were provided by the aureus possesses a wide variety of virulence factors, including Laboratory of Microbiology at the Teaching Hospital of Botu- peptidoglycan, teichoic acid, enzymes and toxins, adhesion 4 catu Medical School, UNESP - Univ Estadual Paulista (City of molecules, and four types of cytotoxins. They can cause Botucatu, São Paulo State, Brazil). different types of infection, many of them acquired in the hos- pital environment. About 20% to 30% of the human population Sample collection is colonized with this bacterium and the nasal mucosa is the 5 main ecological niche. The isolates included in the study were obtained on the A relevant factor that complicates treatment of staphylo- day following growth detection in a Bactec 9120 system and coccal infections is the high resistance rate to ␤-lactam antibi- after confirmation of containing probable Staphylococcus spp. otics observed among Staphylococcus spp., which requires by Gram staining (Gram-positive cocci) and positive catalase large-scale use of expensive or toxic antibiotics. Staphylococ- tests. cus spp. resistance to ␤-lactam antibiotics is mainly due to The blood culture bottles were disinfected with 70% alco- two different mechanisms which, however, can interact. The hol and 3 mL of blood was collected with a 5 mL syringe and first mechanism consists of the production of ␤-lactamase, 70 × 25-mm needle into two microtubes (1.5 mL per micro- an enzyme that hydrolyzes the antibiotic. The second mech- tube). One microtube was used for seeding onto blood agar for anism is related to a change in the site of action of ␤-lactams phenotypic identification and the other microtube was used by producing a new penicillin-binding protein, PBP2a, which for extraction of bacterial DNA for multiplex PCR. has low affinity for antibiotics and is absent in suscepti- 6 ble staphylococci. PBP2a is encoded by the mecA gene. This gene is located on a mobile genetic element called SCCmec Phenotypic identification of S. aureus and (staphylococcal cassette chromosome mec), which is widely coagulase-negative staphylococci 7 distributed among staphylococci. PBP2a acts as a transpep- tidase that resumes cell wall synthesis functions when other Phenotypic identification was performed at the Laboratory PBPs are inhibited, thus ensuring the integrity of the bacterial of Bacteriology, Department of Microbiology and Immunol- 8 ␤ cell in the presence of -lactams. ogy, Botucatu Biosciences Institute, UNESP. The samples were ◦ In view of the above considerations, rapid detection of bac- seeded into blood agar and incubated for 24 h at 37 C. After teremia and the subsequent rapid identification of the bacteria isolation from blood agar, the bacteria were submitted to involved and their susceptibility to antibiotics are of great Gram staining for confirmation of morphological staining b r a z j i n f e c t d i s . 2 0 1 8;2 2(2):99–105 101 characteristics, catalase test for characterization of the genus Table 1 – Primers used for the detection of Staphylococcus Staphylococcus, and coagulase test for differentiation between spp., S. aureus and the mecA gene by multiplex PCR. S. aureus and CoNS. Gene Primer Amplicon(bp) 24 DNA extraction directly from blood culture S. aureus SAU327 – GGA CGA CAT 1250 TAG ACG AAT CA SAU1645 – CGG GCA Before DNA extraction, the samples were treated to remove CCT ATT TTC TAT CT interfering elements in the blood culture. The sample was cen- 25 9 × trifuged at 850 g for 2 min. The supernatant was transferred 16S rRNA 16S1 – 5 CCTATAA- 791 GACTGGGATAACTTCGGG to another microtube and again centrifuged at 11,000 × g for 3 1 min. The supernatant was carefully discarded, leaving only 16S2 – 3 the pellet. Five hundred microliters of lysozyme buffer (1 M CTTTGAGTTTCAAC- EDTA, 0.1 M NaCl, 10 mM Tris, pH 8.0, and 5% Triton X-100) CTTGCGGTCG was then added to the pellet and the mixture homogenized 5 ␮ in a vortex. Next, 800 L benzyl alcohol (Sigma) was added, 26 Coa COA1 – 5 GTA GAT TGG 117 the mixture homogenized in a vortex, and the microtube GCA ATT ACATTT TGG 10 × centrifuged at 7000 g for 5 min. Tw o phases formed in AGG 3 the microtube, the lower phase contained the alcohol with COA2 – 5 CGC ATCAGC interfering substances (inhibitors) and the upper phase the TTT GTT ATC CCA TGT bacteria. A 3 27 ␮ For DNA extraction, 320 L of the liquid was removed from mecA MRS1 – 5 AAAATC- 533 the upper phase and transferred to another microtube and GATGGTAAAGGTTGGC 3 10 ␮L lysozyme was added (10 mg lysozyme in 1 mL 10 mM MRS2 – 3 AGTTCT- Tris–HCl). The microtube was homogenized in a vortex and GCAGTACCGGATTTGC incubated at room temperature for 15 min, homogenizing the 5 microtube at five-minute intervals. After this period, 10 ␮L proteinase K was added and the mixture was incubated for ◦ 15 min at 56 C, homogenizing the microtube at five-minute intervals in a vortex.
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