JCM Accepts, published online ahead of print on 14 May 2014 J. Clin. Microbiol. doi:10.1128/JCM.00918-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Multiple PCR for the identification of six different Staphylococcus spp. and

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simultaneous detection of methicillin and mupirocin resistance

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Campos-Peña, E.1, Martín-Nuñez, E.1, Pulido-Reyes, G.1⎯, Martín-Padrón, J.1φ, Caro-Carrillo, E.1, Donate-

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Correa, J.1, Lorenzo-Castrillejo, I.1, Alcoba-Flórez, J. 1, 2, Machín, F. 1, Méndez-Alvarez, S. 1, 3 *

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1 Unidad

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SantaCruz de Tenerife, Spain. 3Departamento de Microbiología, Universidad de La Laguna, 38206 La Laguna,

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Spain

de Investigación, 2 Servicio de Microbiología, Hospital Universitario Ntra. Sra. de Candelaria, 38010

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⎯ Current address: Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de

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Madrid, Cantoblanco, 28049 Madrid, Spain

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φ Current address: Abbott Nutrition Granada. ANR&D Granada USP. Campus de la Salud.

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venidade la Innovación. Edificio BIC.18016. Granada, Spain

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Runing title: Multiple PCR for identification of Staphylococcus spp.

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∗ Corresponding Author: Sebastián Méndez-Álvarez, M.Sc., Ph.D.

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Grupo de Microbiología Molecular

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Unidad de Investigación

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Hospital Universitario Ntra. Sra. de Candelaria

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Ctra. del Rosario 145

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Santa Cruz de Tenerife

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38010 SPAIN

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Tfn.: 34-922600080 FAX: 34-922600562

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e-mail: [email protected]

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ABSTRACT

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We describe a new efficient, sensitive and fast single tube multiple PCR protocol for

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the identification of the most clinically significant Staphylococcus spp. and the simultaneous

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detection of the methicillin and mupirocin resistance loci. The protocol identifies at the

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species level isolates belonging to S. aureus, S. epidermidis, S.haemolyticus, S. hominis,

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S. lugdunensis or S. saprophyticus.

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Humans are the main natural reservoir of the Gram-positive coagulase-positive

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bacterium Staphylococcus aureus (1). The continuous accumulation of resistance and

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virulence factors in this species has resulted in a worldwide health concern due to an

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associated increase in morbidity and mortality (1, 2, 3). Hospital infections by this agent are

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particularly concerning; especially in those patients who are at risk for complications (i.e.,

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immune-compromised patients, pregnant women, new-borns and babies, cancer patients,

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individuals at dialysis programmes, transplant recipients, etc.) (4). Several methicillin-

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resistant S. aureus (MRSA) clones constitute a global alarm, often being epidemic or even

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a cause of pandemics (5, 6). However, within the genus Staphylococcus not only that

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species constitutes a worrisome pathogen. Thus, several coagulase-negative members of

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the genus are the aetiological agents of diverse hospital-acquired severe infections. The

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most clinically significant examples are the species S. epidermidis, S. saprophyticus, S.

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lugdunensis, S. haemolyticus and S. hominis (7 - 13). Their pathogenic features can

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become so hazardous that new proficient antibiotics and efficient, sensitive and fast

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diagnostic methods constitute cornerstones in the fight against these adverse bacteria (12 -

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15).

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Introduction of novel drugs has always been followed by the prompt appearance of

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new staphylococcal resistances. Methicillin was introduced in 1959 to overcome the

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problems that arose from the increasing prevalence of penicillin-resistant S. aureus isolates

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(16). Two years later, methicillin resistant Staphylococcus aureus (MRSA) strains were

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detected. Few antibiotics are still active against MRSA, being mupirocin one of them.

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Mupirocin is normally used as a topical agent to prevent MRSA invasion (17, 18).

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Moreover, it is also an advisable antibiotic to apply when invasive surgeries are employed

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(17). Unfortunately, two years after its introduction, high-level mupirocin resistance

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appeared and has worryingly increased since. Such resistance is commonly mediated by a

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conjugative plasmid-associated locus (ileS-2) (19). Genetic transfer of ileS-2-plasmids has

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given

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Staphylococcus species (20). The growing incidence of staphylococcal resistant strains has

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made necessary the availability of Staphylococcus spp. identification methods able to

rise

to

mupirocin

resistant

Staphylococcus

clones

belonging

to

several

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simultaneously detect antibiotic resistance, as the herein described new multiple PCR

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protocol.

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Bacterial isolates, identification and susceptibility testing. A total of 67 clinical

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isolates were included in this study for the validation of the assay. Initially, 16 isolates were

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used to test all PCR primers pairs. All these isolates were recovered from clinical samples

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from 67 patients at the Microbiology Service of Hospital Universitario Nuestra Señora de

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Candelaria (HUNSC). Three reference S. aureus strains (ATCC 29213, ATCC 25923,

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NCTC8325), were included in the study as well. Prior to the molecular analysis, all isolates

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were biochemically identified at HUNSC Microbiology Service as follows. Clinical isolates

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were recovered by culturing clinical samples onto Columbia agar plates with 5% sheep

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blood and onto manitol-salt agar (MSA) plates (bio Merieux, Marcy l´Etoile, France). Plates

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were incubated at 35-37 ºC for 24-48 h under aerobic conditions. Phenotypic identification

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of the isolates was done by colony morphology, growth features onto MSA, Gram staining,

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and catalase, coagulase and DNAse tests. Susceptibility testing was performed at

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MSHUNSC according to CLSI criteria (Clinical and Laboratory Standards Institute, U.S.A.).

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Staphylococcus isolates were analyzed by Vitek2 (GPS-511 card) (bio Merieux, Marcy

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l´Etoile, France). Besides, their susceptibility against oxacillin and mupirocin was re-tested

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at the HUNSC Research Unit before proceeding with the molecular analysis. Methicillin

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resistance was confirmed by 1 µg oxacillin disk diffusion testing, using Mueller-Hinton agar

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(DIFCO Laboratories, MI, USA). Intermediate methicillin resistance was confirmed by

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oxacillin E-test strips (AB Biodisk). Mupirocin resistance was screened by the disk diffusion

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method (Oxoid, Basingstoke, England): 5-μg mupirocin disks were used to detect low-level

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resistance, and 200-μg disks to detect high-level resistance. Lastly, confirmation of high-

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level resistance was performed with E-test strips (AB Biodisk, bio Merieux, Marcy l´Etoile,

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France), which yielded the exact MIC for every highly mupirocin-resistant isolate (MIC

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≥ 1,024 μg/ml).

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Molecular biology analyses. The first step in the design of the mPCR was to

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select a variety of specific genes to identify at the species-level clinical isolates from the six

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different staphylococcal taxa, as well as to detect high-resistance to methicillin and/or

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mupirocin. The partially amplified loci are showed in Table 1. The primers selected for

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these amplifications had been previously described and were obtained from a commercial

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source (Integrated DNA Technologies, CA, USA). For development of the mPCR, a DNA

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suspension from each isolate was rapidly prepared as previously described (19). Each

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primers pair was individually tested in a single PCR reaction to ensure they amplify the

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expected band (Figure 1). Each of these single reactions was performed twice using DNA

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suspensions from two different isolates for each species. Moreover, some of the single

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PCRs have been used by us with collections of more than 200 S. aureus, more than 100 S.

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lugdunensis and more than 50 S. saprophyticus isolates (unpublished data). The

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reproducible success we obtained using these primers made us to choose them for

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developing the herein described mPCR. Furthermore, we expected that different pairs

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would yield fragments with different sizes (Figure 1 and Table 1), which facilitate their

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identification after the mPCR. Thus, each band was purified (QIAgen Kit, CA, USA) and

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sequenced in order to confirm their identities by comparison to the NCBI (National Center

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for Biotechnology Information, MD,USA) Data Bank sequences. Sequencing of the

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amplicons was performed on an ABI-PRISM310 genetic analyzer (Applied Biosystems

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Japan Co. Ltd., Tokyo) with BigDyefluorecent terminator chemistry (Applied Biosystems,

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Warrington, United Kingdom). In the case of the S. hominis isolate, the low prevalence of

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mecA-positive S. hominis clinical infections in this hospital, suggested the convenience of a

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molecular identification by sequencing its 16S rDNA. The S. hominis isolate 16S rDNA

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sequence had 99.9% identity with the S. hominis ATCC 27844 16S rRNA gene sequence

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(GenBank: L37601.1). After band identity confirmation, mPCR assay was optimized (Figure

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2) and the working protocol we used is described below: in a 25 μl reaction volume, 2.5μl of

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a DNA suspension was used as DNA template, and it was added to a 22.5μl PCR mixture

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consisting of 1X reaction buffer, 0.2 mM of each of the four dNTPs, 2.4 mM MgCl2, 1 μM of

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nucA primer pair, 0.5 μM of mvaA pair, 0.5 μM of sep pair, 0.5 μM of fbl pair, 0.5 μM of sap

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pair, 0.5 μM of mecA pair and 0.5 μM of ileS2 pair, 0.5 μM of hom pair and 0.1 U/μl of Taq

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DNA polymerase (BiothermTM DNA Polymerase, Gene Craft, Germany). All mPCR assays

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were carried out with a negative control containing all reagents but the DNA template. DNA

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amplification was carried out in a GeneAmp PCR system 9700 thermocycler (PE Applied

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Biosystems, CA, USA) with the following thermal cycling conditions: initial denaturizing step

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at 94ºC for 5 min, followed by 45 amplification cycles: (i) 10 cycles (denaturizing at 94ºC for

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30 s, annealing at 64ºC for 45 s, and extension at 72ºC for 45 s), (ii) 10 cycles (denaturizing

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at 94ºC for 30 s, annealing at 55ºC for 45 s, and extension at 72ºC for 1 min) and (iii) 25

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cycles (denaturizing at 94ºC for 45 s, annealing at 52ºC for 45 s, and extension at 72ºC for

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60 s) ending with a final extension step at 72ºC for 10 min. After the mPCR, 4 μl from the

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reaction tube were subjected to an agarose gel electrophoresis (2% agarose, 1X Tris-

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borate-EDTA, 8.5 V/cm, 75 min), using a 100 bp molecular size standard ladder (Roche,

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Basel, Switzerland) to estimate the sizes of the amplification products. The gel was stained

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with ethidium bromide, and the amplicons were visualized using an UV light in a GelDoc

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System (BIORAD, CA, USA).

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Method Comparison Studies. The concordance, efficiency, reproducibility,

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sensitivity, typeability and the discrimination power of the mPCR, were estimated by use of

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bivariate ratios and the Simpson diversity index. Moreover, combined-comparative

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analyses of gel images and phenotypic data were performed by using the INFOQUEST

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FINGERPRINTING SYSTEM Version 4.5 (BIORAD, CA, USA).

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Results. After performing the mPCR in the 67 isolates, the nucA fragment only

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amplified in S. aureus strains and never in other staphylococcal isolates. Similarly, fbl,

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mvaA, sap, sep and hom fragments only yielded fragments in S. lugdunensis, S.

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haemolyticus, S. saprophyticus, S. epidermidis and S. hominis strains, respectively. As for

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the mecA fragment, it was detected in all strains which exhibited high methicillin resistance

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but not in the sensitive ones. Similarly, amplification of the ileS-2 target always occurred for

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high mupirocin-resistant strains and never for low- or intermediate-resistant, and also never

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for the susceptible ones. The mPCR results for the isolates tested in this study are shown

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in Table 2.

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After analyzing the 67 Staphylococcus spp. isolates by phenotypic, biochemical and

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microbiological tools, single PCRs and by the new described mPCR, concordance of

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classical identification methods with mPCR identification had a value of 1 (100%); a

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sensitivity value of 1 (100%); as well as a specificity value 1 (100%); a typeability value of 1

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(100%); a reproducibility value ≥ 0.95 and a discriminatory power of 0.9225

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(http://insilico.ehu.es/mini_tools/discriminatory_power/index.php).

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Concluding remarks. The clinical infections referred herein constitute health

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concerns and therefore a prompt identification of the staphylococcal infectious agent and

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the precise detection of their antibiotic resistances are crucial for a successful management

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(7 – 9, 18 - 20). With these aims we have developed a new single tube multiple PCR which

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is very fast, extremely efficient and sensitive. A limitation of this multiplex assay is that it

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only can identify S. aureus and the five mentioned coagulase-negative staphylococcal

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species. However, according to the literature (Murray) we have included the most clinically

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significant Staphylococcus spp., which are the most frequently isolated in our hospital as

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well. Other species, as e.g. Staphylococcus schleiferi has been rarely associated to

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endophtalmitis after surgery or Staphylococcus capitis to neonatal sepsis in some studies

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(17) but they have not caused complications in our hospital. Other data that could be

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considered a limitation in this study is the low number of methicillin and/or mupirocin

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resistant isolates we tested. But, as we have mentioned above, the bigger number of

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isolates analysed by single PCRs reinforces the trustworthy value of this multiplex PCR. In

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comparison with good previously described methods, as e.g. the Quadriplex PCR

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described by Zhang et al (13), this new protocol brings the advantage of obtaining species

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specific amplicons, which permits species identification without the need of sequencing

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PCR fragments after the PCR reaction. This protocol, from the preparation of cellular

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suspension to electrophoresis analysis of the PCR products on agarose gel, was performed

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in 5 - 6 h. The knowledge gave by the results obtained should mandate the appropriate

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antibiotic therapy in concert with preemptive measurements.

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Acknowledgements

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This work was partially supported by grants from INSTITUTO DE SALUD CARLOSIII

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(Spanish Health Ministry): PS10/00125 to S.M.A; and PS12/00280 to F.M. We also thank

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cofounding by the European Region Development Funds (ERDF).

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Agents Chemother. 55: 3649-60.

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10. Otto M. Staphylococcus epidermidis—the ‘accidental’ pathogen. 2009. Nat. Rev. Microbiol. 7: 555–67.

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11. Pereira EM, Oliveira FL, Schuenck RP, Zoletti GO, Dos Santos KR. 2010. Detection of

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Staphylococcus lugdunensis by a new species-specific PCR bassed on the fbl gene.

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simultaneous identification of S. aureus and detection of methicillin and mupirocin

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resistance. J. Clin. Microbiol 39: 4037-41.

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FIGURE LEGENDS

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Figure 1. Agarose gel electrophoresis patterns showing single PCR amplification products

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for S. lugdunensis gene fbl (lane 1), mupirocin resistant gene ileS2 (lane 2), S.

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saprophyticus gene sap (lane 3), S. aureus gene nucA (lane 4), methicillin resistant gene

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mecA (lane 5), S. haemolyticus gene mvaA (lane 6), S. epidermidis gene sep (lane 7) and

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S. hominis gene hom (lane 8). Each pair of primers was amplified together with a negative

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control without DNA.

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Figure 2. Agarose gel electrophoresis patterns showing mPCR amplifications products

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from different Staphyloccocal isolates. C-: negative control without DNA; lane 1: methicillin-

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resistant S. hominis isolate (hom and mecA bands); lane 2: methicillin-resistant S.

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epidermidis isolate (sep and mecA bands); lane 3: mupirocin-resistant S. epidermidis

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isolate (sep and ileS2 bands); lane 4: methicillin- and mupirocin resistant S. haemolyticus

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isolate (mvaA, mecA and ileS2 bands); lane 5: methicillin-resistant S. aureus isolate (nucA

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and mecA bands); lane 6: methicillin- and mupirocine-susceptible S. saprophyticus isolate

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(sap band); lane 7: methicillin- and mupirocine-susceptible S. lugdunensis (fbl band).

TABLE 1. Primers used in this study. Target identification (locus)

Primers

S. lugdunensis (fbl)

fblF fblR ileS2F(mup1) ileS2R(mup2) sapF sapR nucF nucR mecA1 mecA2 mvaA1 (hae1) mvaA2 (hae2) sepF sepR homF homR

Mupirocine resistance (ileS2) S. saprophyticus (sap) S. aureus (nuc) Methicillin resistance (mecA) S. haemolyticus (mvaA) S. epidermidis (sep) S. hominis (hom) a

Optimal annealing temperature.

b

Sequence (5’-3’) AAA TCT CCA AGT TGA CCA AAC ATA C GAT TGC GCT GAA AGA ATT GC TAT ATT ATG CGA TGG AAG GTT GG AAT AAA ATC AGC TGG AAA GTG TTG AAC GGG CGT CTC GAT AGA AAA AAC GGG CGT CCA CAA AAT CA TCG CTT GCT ATG ATT GTG G GCC AAT GTT CTA CCA TAG C GTA GAA ATG ACT GAA CGT CCG ATA A CCA ATT CCA CAT TGT TTC GGT CTA A GGT CGC TTA GTC GGA ACA AT CAC GAG CAA TCT CAT CAC CT CAG TTA TAC GGT ATG AGA GC CTG TAG AGT GAC AGT TTG GT TAC AGG GCC ATT TAA AGA CG GTT TCT GGT GTA TCA ACA CC

a

Tm

Amplicon size (bp)

52.6 52.6 52.5 52.5 56.4 57.7 52.9 50.8 54.6 55.7 54.9 54.7 50.2 51.7 52.5 51.1

b

550 456 380 359 310 271 219 177

Reference [Pereira, 2010] [Pérez-Roth, 2001] [Martineau, 2000] [Hirotaki, 2011] [Pérez-Roth, 2011] [Schuenck, 2011] [Hirotaki, 2011] [Hirotaki, 2011]

Base pairs.

TABLE 2. Staphylococcal clinical isolates, identification at the species level, susceptibilities to methicillin and mupirocin and mPCR results. Species S. aureus S. epidermidis S. hominis S. saprophyticus S. haemolyticus S. lugdunensis Total a

Mupirocine.

b

a

Isolates

Mup R

31 14 1 12 3 6 67

2 3 0 1 0 0 6

c

d

Methicillin. Resistant.

c

b

d

Met R

Mup R/Met R

Mup S /Met S

19 2 1 1 1 0 24

5 0 0 0 2 0 7

5 9 0 10 0 6 30

Susceptible