Juana V. Martín-López1,4
Simultaneous PCR detection
Oscar Díez-Gil2
Manuel Morales3
of ica cluster and methicillin
Ninivé Batista2
and mupirocin resistance
Jesús Villar1
Félix Claverie-Martín1
genes in catheter-isolated
Sebastián Méndez-
Summary. Recent data show that more than 50% of catheter-associated blood-
stream infections are caused by staphylococci. Staphylococcal infections produced by intercellular-adhesion cluster (ica) carriers can be even more problematic due to the presence of methicillin and mupirocin resistance genes. In the present study, a multiplex PCR protocol that allows the simultaneous identification of staphylococ- ci and detection of both the ica and methicillin and/or mupirocin resistance genes was developed. Furthermore, the method allows differential detection of the ica locus from Staphylococcus aureus and Staphylococcus epidermidis. [Int
Microbiol 2004; 7(1):63–66]
Key words: Staphylococcus aureus · Staphylococcus epidermidis · intercellular
adhesion gene cluster · antibiotic resistance · multiplex PCR Received 9 April 3003Accepted 13 May 2003 *Corresponding author: Sebastián Méndez-ÁlvarezLaboratorio de Biología MolecularUnidad de InvestigaciónHospital Universitario Ntra. Sra. de CandelariaCtra. del Rosario, s/n38010 Santa Cruz de Tenerife, SpainTel. +34-922600080. Fax +34-922600562E-mail: [email protected] such as catheters [6,7], poses particular problems. These bac- Introduction
teria develop a highly consolidated structure: the biofilm[6,7,25]. In the biofilm, microbial populations reside within a A major achievement within the medical field in the twenti- matrix that facilitates cell-to-cell and/or cell-surface adher- eth century was the invention and development of protesic ence, resulting in an inherent structural resistance towards surgical implants. However, such implants frequently antimicrobial agents, such as antibiotics, disinfectants, and become infected by bacteria [7,18,23]. This is a serious com- germicides [7,8,19,24]. In the case of staphylococci, forma- plication, especially when the infection is caused by multire- tion of the biofilm requires polysaccharidic intercellular sistant bacteria, which are difficult to eradicate from the adhesin (PIA), which is synthesized by enzymes encoded by protesic material. Additionally, the efficiency of some bacte- the intercellular adhesion cluster (ica) [5,11,14].
ria in their ability to colonize indwelling medical devices, Recent data show that 50% of pathogens isolated from hospital-acquired bloodstream infections, normally associ- antibiotic-resistance patterns were determined according to the standard lab- ated with the use of central-venous catheters, are staphylo- oratory criteria of the Microbiology Service of our hospital. S. aureus 229 andS. epidermidis 1855-25 and 9295-79 were methicillin and mupirocin resist- cocci [3,4,9]. Furthermore, many Staphylococcus aureus and ant, whereas S. epidermidis 9295-79 was mupirocin and methicillin sensitive.
Staphylococcus epidermidis strains carry the ica cluster.
The DNA extraction method has been described previously [20,21].
Staphylococcal infections produced by ica carriers can, in After overnight culture on brain-heart infusion agar plates, one or twocolonies (one from a S. aureus isolate and one from a S. epidermidis isolate) turn, be even more problematic due to the presence of methi- were suspended in 20 ml of sterile distilled water, and the suspension was cillin and mupirocin resistance genes [10,15,20].
then heated at 100°C for 20 min. From this suspension, a 5-ml aliquot was Nosocomial infections due to methicillin-resistant directly used as template for PCR amplification.
staphylococci have increased subsequent to the widespreaduse of β-lactamic antibiotics. The transmission of resistant Multiplex PCR amplification. The oligonucleotide primers have all
been previously described [1,10,13,17,20]. icaAB-F: 5'-TTATCAATGC-
strains among hospital departments, and the ability of bacte- CGCAGTTGTC-3' and icaAB-R: 5'-GTTTAACGCGAGTGCGCTAT-3' ria to transmit resistance genes are well-known [18,22]. With from partial icaAB genes of S. epidermidis, icaA-S: 5'-AAACTTGGTGCG- respect to mupirocin, several recent studies indicate the util- GTTACAGG-3' and icaA-E: 5'-TCT GGGCTTGACCATGTTG-3' from partial icaAB genes of S. aureus, FemB1: 5'-TTACAGAGTTAACTGT- ity of topical mupirocin application in order to avoid the col- TACC-3' and FemB2: 5'-ATACAAATCCAGCACGCTCT-3' from femB, onization of indwelling medical devices. While mupirocin is MupA: 5'-TATATTATGCGATGGAAGGTTGG-3' and MupB: 5'- not an antibiotic for treatment of infections once they occur, AATAAAATCAGCTGGAAAGTGTTG-3' from ileS-2 and MecA1: 5'-GTAGAAATGACTGAACGTCCGATAA-3' and MecA2: 5'-CCAATTC- it is used prophylactically to prevent S. aureus strains from CACATTGTTTCGGTCTAA-3' from mecA. Multiplex PCR assays were all reaching the inner part of the device. However, this applica- carried out directly using the bacterial suspension obtained after the rapid tion has led to the selection of highly mupirocin-resistant DNA extraction method described above. A 5-ml aliquot of this suspension staphylococci [2,4,16]. For example, in our hospital, the inci- was added to 45 ml of PCR mixture consisting of 1× reaction buffer [16 mM(NH ) SO , 67 mM Tris-HCl (pH 8.8)], 0.2 mM of each of the four dNTPs dence of mupirocin resistance has increased sharply from (Promega, Madison, WI, USA), 3.5 mM MgCl 3.6 µM of each femB primer, 0.2 µM of each ileS-2 primer, 0.1 µM of each mecA primer, and 0.8 µM of Since most S. aureus are ica carriers, the colonization of each icaAB primer and 1.25 U of Taq DNA polymerase (Bioline, UK). Foreach sample, one reaction was done with the femB pair of primers to identi- devices by isolates harboring both resistance genes and adhe- fy S. aureus strains, with the mecA and ileS-2 pairs of primers to detect both sion genes has become a serious problem. Rapid detection of resistance markers, and with the two icaAB pairs of primers to detect the ica the ica locus in hospital staphylococcal isolates, together cluster from S. aureus, S. epidermidis, or simultaneously from both. In orderto reduce the formation of nonspecific extension products, a “hot-start” PCR with simultaneous detection of antibiotic-resistance genes, protocol was used. All multiple PCR assays were carried out with a negative may allow the development of methods to prevent or reduce control containing all of the reagents without DNA template. DNA was the ability of bacterial to invade indwelling medical devices.
amplified in a GeneAmp PCR system 9700 thermocycler (PE, Applied It should be noted that the presence of the ica locus does not Biosystems, CA, USA) with the following thermal cycling profile: an initialdenaturation step at 94°C for 5 min was followed by 10 cycles of amplifica- guarantee its expression; thus, it does not directly reflect tion (denaturation at 94°C for 30 s, annealing at 66°C for 45 s, and extension biofilm formation. However, anticipating and detecting the at 72°C for 60 s); 5 cycles of amplification (denaturation at 94°C for 45 s, possibility of biofilm colonization of catheters before it actu- annealing at 64°C for 45 s, and extension at 72°C for 60 s); and 25 cycles ofamplification (denaturation at 94°C for 45 s, annealing at 50°C for 45 s, and ally occurs would be of great help in preventing infection.
extension at 72°C for 60 s), ending with a final extension step at 72°C for 10 For this reason, we developed a method to simultaneously min. After PCR amplification, 5 µl were removed and subjected to 2% detect ica and methicillin- and mupirocin-resistance markers.
agarose gel electrophoresis to estimate the size of the amplification productsby comparison with a 100-bp molecular size standard ladder (Roche The method described here is a rapid and easy means to ver- Diagnostics, Mannheim, Germany). The gel was stained with ethidium bro- ify that catheters are not being colonized by mupirocin-resist- mide and the amplicons were visualized using a UV light.
ant, methicillin-resistant staphylococci harboring the ica The reaction conditions for the multiplex PCR assay were optimized to cluster. The presence within the hospital of these staphylo- ensure that all of the target gene sequences were satisfactorily amplified. Theprimers used in this study differ in annealing temperatures, which increased cocci would not only make the preventive use of mupirocin the possibility of occurrence of unwanted bands originating from non-spe- useless, it could also lead to an increased risk of infections.
cific amplification. Therefore, both a “hot-start” and three rounds of ampli-fication with different annealing temperatures were carried out. In addition,there is evidence indicating that multiplex PCR with targets that differ wide-ly in size often favors amplification of the shorter target over the longer one, Materials and methods
resulting in different amounts of amplified products [21]. For this reason, inorder to optimize the conditions for the multiplex PCR analysis, different Bacterial isolates, identification, susceptibility testing,
primer concentrations, template DNA preparations, and MgCl concentra- and DNA extraction method. S. epidermidis strains 1855-25, 9295-
tions were assayed. As described above, the final quantities generated opti- 79, and 9951-79 were isolated from catheters obtained from the Oncology mal results were 3.5 mM MgCl 180 pmol of each femB primer, 10 pmol of Service of our hospital (Nuestra Señora de Candelaria University Hospital, each ileS-2 primer, 5 pmol of each mecA primer, 40 pmol of each icaAB Santa Cruz de Tenerife, Spain). S. aureus strain 229 was isolated from the primer and 5 ml of the DNA solution obtained with our DNA extraction sputum of a patient with sepsis. All strains were biochemically identified, and RESISTANCE GENES IN STAPHYLOCOCCUS Fig. 1. Agarose gel electrophoresis patterns
showing single (lanes 3–7) and multiplex
(lanes 8–10) PCR results: lane 3, icaAB
amplicon from Staphylococcus aureus isolate
229; lane 4, femB amplicon from S. aureus
isolate 229; lane 5, icaAB product from
Staphylococcus epidermidis isolate 9951-79;
lane 6 ileS-2 amplicon from S. epidermidis
isolate 9295-79; lane 7, mecA amplicon from
S. epidermidis isolate 9295-79; lane 8, icaAB,
ileS-2 and mecA amplicons from a S. epider-
isolate 1855-25; lane 9, femB, icaAB,
ileS-2 and mecA amplicons from S. aureus
isolate 229; lane 10, femB, ileS-2, mecA and
both icaAB from a mixture of S. aureus and S.
isolates 229 and 9951-79, respec-
tively, simultaneously amplified. Lane 1,
DNA molecular size marker (100-bp ladder).
No bands were present in the negative control
(lane 2). A schematic representation of the
fragments amplified is shown on the left.
less than 6 h. Moreover, it can be used for S. aureus and Results and Discussion
S. epidermidis and for a mixture of both staphylococci.
Nowadays, since few antibiotics, including mupirocin, Prior to optimizing the multiple PCR, the PCR protocol was constitute the last resort against MRSA, and due to the evaluated in order to ensure that it was adequate for the indi- increasing incidence of catheter-associated staphylococcal vidual amplification of all five DNA fragments. Each indi- bloodstream infections, a fast, sensitive laboratory method to vidual amplification yielded a fragment of the expected size, immediately detect multiple-resistant staphylococci harbor- i.e. 750-, 651-, 546-, 456-, and 310-bp, respectively. Figure 1 ing the ica cluster is urgently needed. Although most shows an agarose gel that illustrates results typically obtained S. aureus carry the ica cluster, confirmation of its presence in with the optimized PCR assay. Amplification of icaAB, femB, a particular isolate is a necessary step in preventing coloniza- ileS-2 and mecA targets produced distinct, easily recogniza- tion by potentially biofilm-forming strains. The method ble bands corresponding to their respective molecular size described herein is highly sensitive and specific, fast and fea- (lanes 3–7). The icaAB fragments were clearly differentiated sible, and thus provides a new tool for controlling catheter- when amplified from S. aureus or from S. epidermidis (lanes 8–10); femB was always amplified in the case of S. aureusstrains (lane 9) and never in the case of S. epidermidis (lane Acknowledgements. This work was supported by grants 2001/020 from
the Consejería de Educación, Cultura y Deportes, Canarian Autonomous
8). Results obtained for the mecA and ileS-2 fragments were Government, FIS PI 01/3150 from the Health Spanish Ministry, and Bio in accordance with the resistance pattern of the isolates: 2002/00953 from the Spanish Ministry for Science and Technology. S.M.A.
methicillin- and mupirocin-resistant isolates S. epidermidis was supported by FIS contract 99/3060 (Fondo de Investigación Sanitaria, 1855-25 and S. aureus 229 presented the mecA fragment and showed the ileS-2 marker (lanes 8–10). PCR from mixed S.
229 and S. epidermidis 9951-79 colonies permitted thesimultaneous detection of the five different targets (lane 10).
This protocol, including the rapid extraction of DNA 1. Anthony RM, Connor AM, Power EGM, French GL (1999) Use of the from single colonies and electrophoretic analysis of the polymerase chain reaction for rapid detection of high-level mupirocin amplified products on an agarose gel, was completed in resistance in staphylococci. Eur J Clin Microbiol Infect Dis 18:30–34 2. Berns JS (2003) Infection with antimicrobial-resistance microorgan- vancomycin and rifampicin on methicillin-resistant Staphylococcus isms in dialysis patients. Seminars in Dialysis 16:30–37 aureus biofilms. Lancet 357:40–41 3. Bouza E, Burillo A, Muñoz P (2002) Catheter-related infections: diag- 16. Johson DW, MacGinley R, Kay TD, Hawley CM, Campbell SB, Isbel nosis and intravascular treatment. Clin Microbiol Infect 8:265–274 NM, Hollet P (2002) A randomised controlled trial of topical exit site 4. Centers for Disease Control and Prevention (2002) Safer, healthier mupirocin application in patients with tunnelled, cuffed haemodialysis people. Guidelines for the prevention of intravascular catheter-related catheters. Nephrol Dial Transplant 17:1802–1807 Infections. Mor Mortal Wkly Rep. Recommendations and reports.
17. Kobayashi N, Wu H, Kojima K, Taniguchi K, Urasawa S, Uehara N, Omizu Y, Kishi Y, Yagihashi A, Kurokawa I (1994) Detection of mecA, 5. Cramton SE, Gerke C, Schnell F, Nichols WW, Götz F (1999) The femA and femB genes in clinical strains of staphylococci using poly- intercellular adhesion (ica) locus is present in Staphylococcus merase chain reaction. Epidemiol Infect 113:259–266 aureus and is required for biofilm formation. Infect Immun 18. Livermore D M (2002) Antibiotic resistance in staphylococci. Int J 6. Donlan RM (2001) Biofilm formation: A clinically relevant microbio- 19. Mack D (1999) Molecular mechanisms of Staphylococcus epidermidis logical process. Clin Infect Dis 33:1387–1392 biofilm formation. J Hosp Infect 43:S113–S115 7. Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of 20. Martín-López JV, Pérez-Roth E, Claverie-Martín F, Díez-Gil O, Batista clinically relevant microorganisms. Clin Microbiol Rev 15:167–193 N, Morales M, Méndez-Álvarez S (2002) Detection of Staphylococcus 8. Dunne WM Jr (2002) Bacterial adhesion: seen any good biofilms late- aureus clinical isolates harbouring the ica gene cluster needed for biofilm establishment. J Clin Microbiol 40:1569–1570 9. Eggimann P, Pittet D (2002) Overview of catheter-related infections 21. Pérez-Roth E, Claverie-Martín F, Villar J, Méndez-Álvarez S (2001) with special emphasis on prevention based on educational programs.
Multiplex PCR for simultaneous identification of Staphylococcus aureus and detection of methicillin and mupirocin resistance. J Clin 10. Frebourg NB, Lefebvre S, Baert S, Lemeland JF (2000) PCR-based assay for discrimination between invasive and contaminating 22. Pérez-Roth E, Claverie-Martín F, Batista N, Moreno A, Méndez- Staphylococcus epidermidis strains. J Clin Microbiol 38:877–880 Álvarez S (2002) Mupirocin resistance in methicillin-resistant 11. Galdbart JO, Allignet J, Tung HS, Rydèn C, El Solh N (2000) Staphylococcus aureus clinical isolates in a Spanish hospital. Co-appli- Screening for Staphylococcus epidermidis markers discriminating cation of multiplex PCR assay and conventional microbiology meth- between skin-flora strains and those responsible for infections of joint ods. Diagn Microbiol Infect Dis 43:123–128 23. Raad I, Alrahwan A, Rolston K (1998) Staphylococcus epidermidis: 12. O’Gara J, Humphreys H (2001) Staphylococcus epidermidis biofilms: Emerging resistance and need for alternative agents. Clin Infect Dis importance and implications. J Med Microbiol 50:582–587 13. Geha DJ, Uhl JR, Gustaferro CA, Persing DH (1994) Multiplex PCR 24. Rachid S, Ohlsen K, Witte W, Hacker J, Ziebuhr W (2000) Effect of for identification of methicillin-resistant staphylococci in the clinical Subinhibitory antibiotic concentrations on polysaccharide intercellular laboratory. J Clin Microbiol 32:1768–1772 adhesin expression in biofilm-forming Staphylococcus aureus.
14. Götz F (2002) Staphylococcus and biofilms. Mol Microbiol Antimicrob Agents Chemother 44:3357–3363 25. Stewart PS, Costerton JW (2001) Antibiotic resistance of bacteria in 15. Jones SM, Morgan M, Humphrey TJ, Lappin-Scott H (2001) Effect of Detección simultánea del cluster ica y de los genes de
Detecção simultânea do cluster ica e dos genes de
resistencia a meticilina y mupiricina en Staphylococcus
resistência a meticilina e mupiricina em Staphylococcus
aislados de catéteres
isolados de catéteres
Resumen. Estudios recientes han demostrado que más del 50% de las sep-
Resumo. Estudos recentes têm demonstrado que mais de 50% das sep-
ticemias asociadas al uso de catéteres están causadas por estafilococos. Las ticemias associadas ao uso de catéteres são causadas por estafilococos. As infecciones estafilococales producidas por cepas portadoras del operón de infecções estafilocócicas produzidas por cepas portadoras de operón de adhesión intercelular (ica) pueden agravarse por la presencia de genes de adesão intercelular (ica) podem agravar-se pela presença de genes de resistencia a la meticilina y/o a la mupirocina. Hemos desarrollado un pro- resistência a meticilina e/ou a mupirocina. Foi desenvolvido um protocolo tocolo de PCR múltiple que permite, simultáneamente, identificar estafilo- de PCR múltiplo que permite, simultaneamente, identificar estafilococos e cocos y detectar la presencia de ica y de los genes de resistencia a la meti- detectar a presença de ica e dos genes de resistência a meticilina e/ou a cilina y/o a la mupirocina. Además, dicho método permite la detección dife- mupirocina. Este método permite a detecção diferencial do locus ica de rencial del locus ica de Staphylococcus aureus y/o S. epidermidis. [Int
Staphylococcus aureus e/ou de S. epidermidis. [Int Microbiol 2004;
Microbiol 2004; 7(1):63–66]
Palabras clave: Staphylococcus aureus · Staphylococcus epidermidis ·
Palavras chave: Staphylococcus aureus · Staphylococcus epidermidis ·
cluster génico de adhesión intercelular · resistencia a antibióticos · PCR cluster gênico de adesão intercelular · resistência a antibióticos · PCR multi-

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