Biofilm formation in invasive Staphylococcus aureus isolates is associated with the clonal lineage

doi:10.1016/j.micpath.2015.10.023

Microbial Pathogenesis

Volume 90, January 2016, Pages 41-49

https://doi.org/10.1016/j.micpath.2015.10.023 Get rights and content

Abstract

The contribution of the genetic background of Staphylococcus aureus to biofilm formation is poorly understood. We investigated the association between the genetic background and the biofilm forming ability of clinical invasive S. aureus isolates. Secondary objectives included investigating any correlation with biofilm formation and methicillin resistance or the source of bacteraemia. The study was conducted at a 1300-bed tertiary hospital in Cape Town, South Africa. S. aureus isolates obtained from blood cultures between January 2010 and January 2012 were included. Genotypic characterization was performed by PFGE, spa typing, SCCmec typing and MLST. Thirty genotypically unique strains were assessed for phenotypic biofilm formation with the microtitre plate assay. All isolates were tested in triplicate and an average optical density, measured at a wavelength of 490 nm, was determined. The biofilm forming ability of isolates with A₄₉₀ ≤ 0.17 were considered non-adherent, A₄₉₀ > 0.17 ‘weak positive’ and A₄₉₀ > 0.34 ‘strong positive’. Fifty seven percent of isolates formed biofilms. Weak biofilm formation occurred in 40% (n = 12) and strong biofilm formation in 17% (n = 5) of isolates. All 5 isolates capable of strong biofilm formation belong to one spa clonal complex (spa-CC 064). Strains from spa-CC 064 were capable of higher biofilm formation than other spa clonal complexes (p = 0.00002). These 5 strains belonged to MLST CC5 and CC8. Biofilm formation correlates with the spa clonal lineage in our population of invasive S. aureus strains. Biofilm formation did not correlate with methicillin resistance and was not related to the source of bacteraemia.

Introduction

Staphylococcus aureus has ensured its success as an important pathogen worldwide through its versatility, virulence factors and resistance mechanisms. Invasive S. aureus infections have a high mortality rate and infections with methicillin-resistant S. aureus (MRSA) have even poorer outcomes [1], [2]. Biofilm formation is a major virulence factor of S. aureus.

A biofilm is ‘an assemblage of surface-associated microbial cells that is encased in an extracellular polymeric substance matrix’ [3]. These unique communities form on various indwelling medical devices. Bacteria embedded in a biofilm are more resistant to antimicrobials through several mechanisms. Overall antimicrobial penetration is poor, growth-dependent agents have decreased efficacy due to the slower metabolic state of the bacteria and exchange of resistance genes is easier due to the close proximity of cells. Formation of persister-cells, a subpopulation of bacteria that survive antimicrobial treatment, is also a contributing factor [4].

Due to advances in the medical field, prosthetic devices are increasingly used in patient management. Biofilms may develop on intravascular catheters, prosthetic heart valves and orthopaedic implants. Prosthetic devices become reservoirs for persistent infections and foci for metastatic complications such as endocarditis, deep tissue abscesses, septic arthritis, and osteomyelitis [5], [6]. Definitive management of device-related infection frequently requires removal or replacement of the prosthetic material.

The best-described mechanism of biofilm development in S. aureus involves the extracellular molecule polysaccharide intercellular adhesin or poly-N-acetylglucosamine (PIA/PNAG) [7]. PIA/PNAG synthesis is regulated by the intercellular adhesion (ica) locus [8]. PIA/PNAG and ica-independent biofilm formation, particularly in MRSA, has also been described [9], [10]. However, the relationship between biofilm formation and the genetic background of S. aureus is poorly understood. Different clonal lineages of S. aureus may have different biofilm forming capabilities. In the recent literature, differences in biofilm formation were found to be due to the staphylococcus protein A (spa) lineage [11]. Other studies found strong biofilm formation correlated with multilocus sequence typing (MLST) clonal complexes [12] or Staphylococcal Chromosome Cassette mec (SCCmec) typing [13].

This study investigated the association between the genetic background and the biofilm forming ability of clinical invasive S. aureus isolates. In addition, we investigated any correlation with biofilm formation and methicillin resistance or source of bacteraemia.

Section snippets

Setting & design

This was a prospective, descriptive study conducted at the National Health Laboratory Service (NHLS) Microbiology Laboratory, Tygerberg Hospital, which is a 1300-bed tertiary referral hospital in Cape Town, South Africa.

Bacterial strains

S. aureus isolates obtained from pure blood cultures between January 2010 and January 2012 were included. Positive blood cultures were identified using the BACTEC 9240 system (Becton Dickinson, USA). Identification of S. aureus was done using Mannitol Salt agar (MSA) and DNase

Description of isolates

The 30 isolates selected comprised of 19 PFGE clusters, with 28 spa types and 15 MLST sequence types (ST). These were from 9 spa-clonal complexes (spa-CC) and 9 MLST clonal complexes (MLST CC) (Table 2). There were 9 MRSA (30%) and 21 methicillin-sensitive S. aureus (MSSA) (70%). The source of infection was determined for all the S. aureus bacteraemias (Fig. 1). Ten originated from skin and soft tissue infections (SSTI), 9 from catheter-related blood-stream infections (CRBSI), 3 pneumonias

Discussion

Biofilm-associated infections caused by S. aureus are a significant cause of morbidity and mortality [24]. In this study strong biofilm formation was associated with strains from spa-CC 064 and MLST CC 5 and 8. Although MLST CC8 has now been incorporated into MLST CC5, it is considered separately in this analysis [15]. As MLST CC8 in now incorporated into MLST CC5, MLST CC5 may be associated with high biofilm formation. This suggests that certain clones are more prone to biofilm formation and

Conflict of interest

The authors have no conflicts of interest to declare.

Declaration on laboratory work

Declaration of contribution to laboratory work.

The biofilm assay was performed by Preneshni Naicker.

Clinical data collection and folder reviews were conducted by Preneshni Naicker.

PFGE, MLST, spa typing and SCCmec typing was performed by Karayem Karayem.

Acknowledgements

This study was funded by the National Health Laboratory Service (NHLS) Research Trust. We are also grateful to the staff at NHLS Microbiology Tygerberg Hospital and to Mediclinic for funding laboratory equipment.

References (37)

F.D. Lowy
Staphylococcus aureus infections
N. Engl. J. Med.
(1998)
S.E. Cosgrove et al.
Management of methicillin-resistant Staphylococcus aureus bacteremia
Clin. Infect. Dis.
(2008)
R.M. Donlan
Biofilms: microbial life on surfaces
Emerg. Infect. Dis.
(2002)
S.E. Cramton et al.
Biofilm development in Staphylococcus
J.W. Costerton et al.
Bacterial biofilms: a common cause of persistent infections
Science
(1999)
V.H. Chu et al.
Staphylococcus aureus bacteremia in patients with prosthetic devices: costs and outcomes
Am. J. Med.
(2005)
D. Mack et al.
The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis
J. Bacteriol.
(1996)
S.E. Cramton et al.
The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation
Infect. Immun.
(1999)
F. Fitzpatrick et al.
Evidence for icaADBC-independent biofilm development mechanism in methicillin-resistant Staphylococcus aureus clinical isolates
J. Clin. Microbiol.
(2005)
E. O'Neill et al.
A novel Staphylococcus aureus biofilm phenotype mediated by the fibronectin-binding proteins, FnBPA and FnBPB
J. Bacteriol.
(2008)

S.S. Atshan et al.

Comparative characterisation of genotypically different clones of MRSA in the production of biofilms

J. Biomed. Biotechnol.

(2012)

S. Croes et al.

Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage

BMC Microbiol.

(2009)

Y. Lim et al.

Predictive genetic risk markers for strong biofilm-forming Staphylococcus aureus: fnbB gene and SCCmec type III

Diagn. Microbiol. Infect. Dis.

(2013)

R.H. Deurenberg et al.

The molecular evolution of methicillin-resistant Staphylococcus aureus

Clin. Microbiol. Infect.

(2007)

W.F. Oosthuysen et al.

Population structure analyses of Staphylococcus aureus at Tygerberg Hospital, South Africa, reveals a diverse population, a high prevalence of Panton-Valentine leukocidin genes, and unique local methicillin-resistant S. aureus clones

Clin. Microbiol. Infect.

(2013)

L.K. McDougal et al.

Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database

J. Clin. Microbiol.

(2003)

D. Harmsen et al.

Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management

J. Clin. Microbiol.

(2003)

C. Milheirico et al.

Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus

Antimicrob. Agents Chemother.

(2007)

Cited by (39)

Alleviating the virulence of Pseudomonas aeruginosa and Staphylococcus aureus by ascorbic acid nanoemulsion
2023, Research in Microbiology
The high incidence of persistent multidrug resistant bacterial infections is a worldwide public health burden. Alternative strategies are required to deal with such issue including the use of drugs with anti-virulence activity. The application of nanotechnology to develop advanced Nano-materials that target quorum sensing regulated virulence factors is an attractive approach. Synthesis of ascorbic acid Nano-emulsion (ASC-NEs) and assessment of its activity in vitro against the virulence factors and its protective ability against pathogenesis as well as the effect against expression of quorum sensing genes of Pseudomonas aeruginosa and Staphylococcus aureus isolates. Ascorbic acid Nano-emulsion was characterized by DLS Zetasizer Technique, Zeta potential; Transmission Electron Microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR). The antibacterial activity of ASC-NEs was tested by the broth microdilution method and the activity of their sub-MIC against the expression of quorum sensing controlled virulence was investigated using phenotypic experiments and RT-PCR. The protective activity of ASC-NEs against P. aeruginosa as well as S. aureus pathogenesis was tested in vivo. Phenotypically, ASC-NEs had strong virulence inhibitory activity against the tested bacteria. The RT-PCR experiment showed that it exhibited significant QS inhibitory activity. The in vivo results showed that ASC-NEs protected against staphylococcal infection, however, it failed to protect mice against Pseudomonal infection. These results suggest the promising use of nanoformulations against virulence factors in multidrug resistant P. aeruginosa and S. aureus. However, further studies are required concerning the potential toxicity, clearance and phamacokinetics of the nanoformulations.
Staphylococcus aureus surface protein G (sasG) allelic variants: correlation between biofilm formation and their prevalence in methicillin-resistant S. aureus (MRSA) clones
2022, Research in Microbiology
Citation Excerpt :
The endemic CC5 and CC8 MRSA clones, which are the most frequent in bloodstream infections in Spain [14], developed stronger biofilms than the sporadic isolates, such as CC1, CC30, CC45, CC72, CC88, and CC398. Previous studies have supported this observation by demonstrating that the CC5 and CC8 strains form stronger biofilms than strains from other clonal complexes [22–25]. This suggests that the ability to form strong biofilms in endemic clones may be an important virulence factor, helping the bacteria survive in the host or in the hospital environment and transforming them into successful pathogens.
Methicillin-resistant Staphylococcus aureus (MRSA) may persist for long periods due to biofilm formation. The objective of this study was to describe biofilm formation in association with the presence of S. aureus surface protein G (sasG) and its allelic variants in MRSA bacteraemia isolates from endemic (CC5, CC8, CC22) and sporadic clones in Spain (2008–2015). Crystal violet staining was used to assess biofilm formation; DNA microarray, RT-qPCR, and long-read whole genome sequencing were applied to determine the presence, expression and structure of sasG, respectively. The endemic CC5 and CC8 clones produced more biofilm than the sporadic clones; these endemic clones carried sasG allelic variant 1. Otherwise, sporadic clones, with less biofilm formation, showed either an absence of sasG (65%) or the presence of allelic variant 2 (35%). Variants 1 and 2 differed in the expression of sasG (1.56 ± 1.20 and 0.37 ± 0.32, respectively). The analysis of a large cohort of closed S. aureus genomes available on the NCBI database confirmed the distribution of the two allelic variants with low amino acid identity (68.1%) among endemic and sporadic clones. SasG variant 1 present in the major CC5 and CC8 clones was correlated with increased biofilm formation and may represent an important virulence determinant.
Production of biofilm by Staphylococcus aureus: Association with infective endocarditis?
2021, Enfermedades Infecciosas y Microbiologia Clinica
Citation Excerpt :
Moreover, Fernández-Hidalgo et al. only demonstrated an association between biomass production and the clonal linage of 209 S. aureus strains causing IE, whereas no other relationship was found between biomass production and other prognostic characteristics.9 Similar findings were also reported by Naicker et al., who could only demonstrate an association between biofilm formation and the clonal linage of S. aureus isolates, but not between biofilm formation and the source of bacteremia.15 These data correlate with our study findings, which show that neither high biomass production nor high metabolic activity of S. aureus strains was associated with IE or C-RBSI.
Staphylococcus aureus is a well-known biofilm-producing pathogen that is capable of causing chronic infections owing to its ability to resist antibiotic treatment and obstruct the immune response. However, the possible association between high biofilm production and infective endocarditis (IE) has not been assessed. Our objective was to compare production of biofilm by S. aureus strains isolated from patients with bacteremia and IE, catheter-related bloodstream infection (C-RBSI), or non-device associated bacteremia.
We isolated 260 S. aureus strains from the blood of patients with bacteremia who were diagnosed during hospital admission between 2012 and 2015. Patients were divided into 3 groups according to whether they had IE, C-RBSI, or non-device associated bacteremia. Biofilm production was measured in terms of biomass and metabolic activity using the crystal violet and XTT assays, respectively. High biomass and metabolic activity rates (based on tertile ranks classification) were compared between the 3 groups.
The high biomass and metabolic activity rates of each group were 41.9% and 37.2% for IE, 32.5% and 35.0%, for C-RBSI, and 29.0% and 33.3% for non-device associated bacteremia (p = 0.325 and p = 0.885, respectively).
High biomass and metabolic activity levels for S. aureus isolates from IE were similar to those of S. aureus isolates from C-RBSI or non-device associated bacteremia.
Staphylococcus aureus es un conocido microorganismo productor de biofilm, capaz de causar infecciones crónicas debido a su capacidad de resistir el tratamiento antibiótico y dificultar la respuesta inmunitaria. Sin embargo, no se ha evaluado la posible asociación entre una elevada producción de biofilm y la endocarditis infecciosa (EI). Nuestro objetivo fue comparar la producción de biofilm por parte de cepas de S. aureus aisladas de pacientes con bacteriemia y EI, bacteriemia relacionada con el catéter (BRC) o bacteriemia no asociada a dispositivos.
Se aislaron 260 cepas de S. aureus de sangre de pacientes con bacteriemia que fueron diagnosticados durante su ingreso hospitalario entre 2012 y 2015. Los pacientes se dividieron en tres grupos según tuvieran EI, BRC o bacteriemia no asociada a dispositivos. La producción de biofilm se midió en términos de biomasa y de actividad metabólica utilizando los ensayos de cristal violeta y XTT, respectivamente. Se compararon los índices de alta biomasa y actividad metabólica (basadas en clasificación por terciles) entre los tres grupos.
Los índices altos de biomasa y actividad metabólica de cada grupo fueron del 41,9 y del 37,2% para EI, del 32,5 y del 35,0% para BRC, y del 29,0 y del 33,3% para bacteriemia no asociada a dispositivos (p = 0,325 y p = 0,885, respectivamente).
Los niveles altos de biomasa y actividad metabólica de los aislados de S. aureus procedentes de EI fueron similares a los de los aislados de BRC o de bacteriemia no asociada a dispositivos.
Biofilm formation by Staphylococcus aureus isolated from food poisoning outbreaks and effect of Butia odorata Barb. Rodr. Extract on planktonic and biofilm cells
2020, LWT
Citation Excerpt :
Moreover, Torlak, Korkut, Uncu, and Sener (2017) found that 90.6% of S. aureus isolates had the ability to form biofilm on polystyrene. In another study, 57% of the S. aureus isolates formed biofilm on polystyrene (Naicker, Karayem, Hoek, Harvey, & Wasserman, 2016). Biofilm formation is important for the survival of staphylococci in a food-processing environment.
The aims of this study were to verify the ability of S. aureus isolates from food poisoning outbreaks to form biofilm, and evaluate the susceptibility of these isolates to Butia odorata extract (BOE) and its effect on S. aureus biofilm. All 13 S. aureus isolates formed biofilm on polystyrene; and 84.6% (11/13) carried icaA and icaB genes, 69.2% (9/13) the icaD gene, and 61.5% (8/13) the icaC gene. All S. aureus isolates were susceptible to BOE, with minimum inhibitory concentration (MIC) values ranging from 2.8 to 11.4 mg.mL-¹. Confocal laser scanning microscopy (CLSM) analysis showed that BOE damaged the cell membrane of S. aureus. The addition of 2 X MIC (60 min contact) decreased the number of biofilm cells by 99.99%, and using 4 X MIC (30 and 60 min contact) the reduction was 99.9%. All S. aureus isolates were biofilm formers, which causes concern because these isolates were involved in outbreaks. On the other hand, the isolates were susceptible to BOE, as the extract damaged the cell membrane of S. aureus. Besides that, BOE reduced the number of S. aureus biofilm cells, having the potential to be used as a sanitizer.
Antistaphylococcal activity of 2(5H)-furanone derivatives
2019, New and Future Developments in Microbial Biotechnology and Bioengineering: Microbial Biofilms Current Research and Future Trends in Microbial Biofilms
The formation of the rigid biofilms by Staphylococcus aureus, the leading opportunistic pathogen causing a variety of nosocomial infections, strongly affects the healing of wounds because of the highly increased tolerance of biofilm-embedded bacteria to antimicrobials and elevated secretion of virulence factors. Therefore, the disintegration of biofilms on tissues and surfaces as well as suppression of the growth of the new ones or development of antimicrobials with ability to penetrate efficiently into the biofilm is a great challenge in infectious disease treatment. Among various low molecular weight compounds affecting the biofilm formation, the derivatives of 2(5H)-furanone have been intensively investigated as a potential antibiofilm agents. Most of them have been shown to interfere with autoinducer-1 (AI-1) and autoinducer-2 (AI-2) system of Gram-negative bacteria, a few compounds demonstrated antimicrobial and antibiofilm activities against Staphylococci as well. In this chapter an attempt has been made to describe the state-of-the-art knowledge about antimicrobial and biofilm-preventing activities of halogenated derivatives of 2(5H)-furanone. The mechanism of action and safety of compounds are also discussed.
Association between biomass formation and the prognosis of infective endocarditis due to Staphylococcus aureus
2020, Enfermedades Infecciosas y Microbiologia Clinica
Citation Excerpt :
However, none of these complexes was associated with mortality or complications in the previous clinical study.2 These results are consistent with those of another study of clinically invasive isolates, in which CC5 proved to be a strong biofilm producer.8 Conversely, CC8 and CC15, which were clonal complexes that produced fewer biomass, were respectively associated with higher in-hospital mortality and higher early mortality (≤2 days).
The aim of this study was to evaluate the association between biomass formation and the clinical characteristics and prognosis of Staphylococcus aureus infective endocarditis (IE).
We prospectively studied 209 S. aureus strains causing IE. Biomass formation was examined using the crystal violet assay and quantified spectrophotometrically. The average (SD) optical density of the biomass was compared for each clinical, microbiological (methicillin-resistance, vancomycin MIC ≥ 1.5 μg/ml) and molecular (clonal complex, agr type and agr dysfunction) variable according to their presence or absence. The primary clinical endpoints studied were in-hospital death, severe sepsis, persistent bacteraemia, symptomatic peripheral embolisms and prosthetic valve IE.
Mean age was 66.1 years, 61.5% of patients were male and the median age-adjusted Charlson comorbidity index was 5 points (IQR 3–8). In-hospital mortality was 37.3%. Strains belonging to CC5 and CC22 had optical biomass densities [mean (SD) 1.573 (1.14) vs 0.942 (0.98) p < 0.001 and 1.720 (0.94) vs 1.028 (1.04) p = 0.001, respectively]. Strains belonging to CC5 and CC22 had significantly higher optical biomass densities [1.369 (1.18) vs 0.920 (0.93) p = 0.008]. No statistically significant differences were found in the clinical endpoints studied.
High biomass production was associated with CC5 and CC22 but not with higher hospital mortality, septic complications, type of endocarditis, methicillin-resistance, elevated vancomycin MIC or agr dysfunction.
La bacteriemia por Staphylococcus aureus es un problema de salud importante asociado a una elevada mortalidad. El objetivo de este estudio fue evaluar la asociación entre la capacidad de formación de biomasa y las características clínicas y el pronóstico de la endocarditis infecciosa (EI) por Staphylococcus aureus.
Se estudiaron de forma prospectiva 209 cepas de S. aureus causantes de episodios de EI. La formación de biomasa se estudió mediante la técnica de cristal violeta y se cuantificó por espectrometría. La media (DE) de la densidad óptica de la biomasa se comparó para cada variable clínica, microbiológica (resistencia a la meticilina, CMI de vancomicina ≥ 1,5 μg/ml) y molecular (complejo clonal, tipo y disfunción de agr) según su presencia o ausencia. El criterio principal de valoración fue la mortalidad hospitalaria. Otras variables clínicas evaluadas fueron: septicemia grave, bacteriemia persistente, embolias periféricas sintomáticas y EI sobre válvula protésica.
La edad media (DE) fue de 66,1 (16,2) años, el 61,5% eran varones y la mediana del índice de comorbilidad de Charlson ajustado a la edad fue de 5 puntos (RIC 3-8). La mortalidad hospitalaria fue del 37,3%. Las cepas pertenecientes a CC5 y CC22 presentaron densidades ópticas de biomasa significativamente más elevadas (media [DE] 1,573 [1,14] frente a 0,942 [0,98] p < 0,001 y 1,720 [0,94] frente a 1,028 [1,04]; p = 0,001, respectivamente). Las cepas pertenecientes a los grupos agrII mostraron mayores densidades ópticas de biomasa (1,369 [1,18] frente a 0,920 [0,93]; p = 0,008). No se observaron diferencias estadísticamente significativas en las variables clínicas estudiadas.
La producción elevada de biomasa se asoció a determinados linajes clonales (CC5 y CC22), pero no se asoció a una mayor mortalidad hospitalaria, complicaciones sépticas, tipo de endocarditis, resistencia a la meticilina, CMI de vancomicina elevada o disfunción del agr.

View all citing articles on Scopus

View full text

Biofilm formation in invasive Staphylococcus aureus isolates is associated with the clonal lineage

Abstract

Introduction

Section snippets

Setting & design

Bacterial strains

Description of isolates

Discussion

Conflict of interest

Declaration on laboratory work

Acknowledgements

Staphylococcus aureus infections

N. Engl. J. Med.

Management of methicillin-resistant Staphylococcus aureus bacteremia

Clin. Infect. Dis.

Biofilms: microbial life on surfaces

Emerg. Infect. Dis.

Biofilm development in Staphylococcus

Bacterial biofilms: a common cause of persistent infections

Science

Staphylococcus aureus bacteremia in patients with prosthetic devices: costs and outcomes

Am. J. Med.

The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis

J. Bacteriol.

The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation

Infect. Immun.

Evidence for icaADBC-independent biofilm development mechanism in methicillin-resistant Staphylococcus aureus clinical isolates

J. Clin. Microbiol.

A novel Staphylococcus aureus biofilm phenotype mediated by the fibronectin-binding proteins, FnBPA and FnBPB

J. Bacteriol.

Comparative characterisation of genotypically different clones of MRSA in the production of biofilms

J. Biomed. Biotechnol.

Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage

BMC Microbiol.

Predictive genetic risk markers for strong biofilm-forming Staphylococcus aureus: fnbB gene and SCCmec type III

Diagn. Microbiol. Infect. Dis.

The molecular evolution of methicillin-resistant Staphylococcus aureus

Clin. Microbiol. Infect.

Population structure analyses of Staphylococcus aureus at Tygerberg Hospital, South Africa, reveals a diverse population, a high prevalence of Panton-Valentine leukocidin genes, and unique local methicillin-resistant S. aureus clones

Clin. Microbiol. Infect.

Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database

J. Clin. Microbiol.

Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management

J. Clin. Microbiol.

Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus

Antimicrob. Agents Chemother.