Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction.

We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.

Genetic assessment of Staphylococcus aureus in an underreported locality: Ambulatory care clinic.

BACKGROUND: Staphylococcus aureus has strong association with anthropogenic environments. This association has not been well supported by use of genetic tools. The aim of this study was to phylogenetically relate numerous isolates from three environments - NCBI samples from hospitals, a community, and a previously unexplored healthcare environment: an ambulatory care clinic (ACC). METHODS: This study incorporated hospital samples from NCBI, a community database from the University of Central Florida (UCF), and newly added samples taken from employees of an ambulatory care clinic located at UCF. Samples were collected from nasal swabs of employees, and positive samples were cultured, extracted, and sequenced at seven MLST loci and one virulence locus (spa). MLST sequences were used in eBURST and TCS population structure analyses and all sequences were incorporated into a phylogenetic reconstruction of relationships. RESULTS: A total of 185 samples were incorporated in this study (15 NCBI sequences from hospital infections, 29 from the ACC, and 141 from the community). In both phylogenetic and population genetics analyses, samples proved to be panmixic, with samples not segregating monophyletically based on sample origin. CONCLUSION: Samples isolated from ambulatory care clinics are not significantly differentiated from either community or hospital samples at the representative loci chosen. These results strengthen previous conclusions that S. aureus may exhibit high genetic similarity across anthropogenic environments.

Assessment of Staphylococcus aureus along milk value chain and its public health importance in Sebeta, central Oromia, Ethiopia.

BACKGROUND: Staphylococcus aureus is one of the leading causes of gastroenteritis acquired from contaminated foods such as milk and milk products. However, such information is limited in Ethiopia. A cross-sectional study was conducted to assess the contamination of milk with S. aureus and knowledge, attitudes and practices (KAP) of actors along the milk value chain in Sebeta, Central Oromia, Ethiopia. A total of 291 samples collected from dairy farms, milk collection centers (MCCs) and processing plant were examined using standard microbiological techniques. The antimicrobial susceptibility profiles of the isolates were also investigated. The KAP of actors in milk value chain were evaluated through a structured questionnaire. RESULTS: Overall, 23.4% (n = 68) of the samples were positive for S. aureus. The prevalence of S. aureus was 19.6% (95% CI: 14.5-25.6) and 80.0% (95% CI: 14.5-25.6) at farm level and MCCs, respectively. Higher isolation rate was observed in the MCCs (p = 0.000) than the farms. The contamination rates of hands of milkers' and milking buckets with S. aureus were 32% and 11.1%, respectively. S. aureus was not isolated from pasteurized milk samples. The isolates were found to be resistant to cefoxitin (100%), penicillin G (98.5%), and streptomycin (77.9%). Among 23 interviewed farmers, 35% of them consumed raw milk, none of them wash their hands and 82.6% did not wash udder and teat before milking. Six percent of consumers had the habit of raw milk consumption. Eighty seven percent of dairy farmers and 54% of consumers had no awareness about milk borne diseases and staphylococcal food poisoning. CONCLUSIONS: The study revealed a high prevalence of S. aureus along the milk value chain, poor milk handling practices, raw milk consumption behavior, lack of awareness about milk borne diseases and occurrence of antimicrobials resistant S. aureus. S. aureus seems to pose a public health risk in Sebeta. Authors recommended the urgent need of public awareness creation about the importance of hygienic milk production and proper handling and adequate heat treatment of milk before consumption and further study to assess cost-effective preventive and control options.

Multiple-locus variable-number tandem repeat fingerprinting as a method for rapid and cost-effective typing of animal-associated Staphylococcus aureus strains from lineages other than sequence type 398.

In veterinary medicine, Staphylococcus aureus is associated with a range of mild to severe infections. The high density of livestock in intensive farming systems increases the risk of disease spread and hampers its control and measures of prevention, making S. aureus one of the most important animal pathogens. Multiple-locus variable-number tandem repeat fingerprinting (MLVF) has been successfully applied to the characterization of livestock-associated meticillin-resistant Staphylococcus aureus (MRSA) ST398 but not to the characterization of a wide range of other animal isolates. The objective of the current study was to examine the effectiveness of MLVF for studying S. aureus strains isolated from households, farms and exotic animals in three regions of Poland. MLVF, random amplification of polymorphic DNA (RAPD), spa typing and diagnostic microarrays were compared to determine the most suitable combination of methods for veterinary purposes. MLVF generated results consistent with host and geographic origins, reflecting population structures with a high concordance to spa typing results. MLVF has been proven to be a rapid, highly discriminatory and cost-effective method suitable for molecular typing in veterinary settings.

New epidemiology of Staphylococcus aureus infections in the Middle East.

Staphylococcus aureus is a bacterial pathogen that is distributed worldwide and represents an increasing problem, both in hospitals and in the community. Global transmission of methicillin-resistant S. aureus (MRSA) has been the subject of many studies. Determining the incidence of colonization with community-acquired MRSA in hospitalized patients and outpatients has been the aim of several studies conducted in the Middle East (western Asia). The local epidemiology within countries in this region is changing, owing to the introduction of new strains with the intercontinental exchange of several clones. Sequence type 80-MRSA-IV is one common clone detected in different countries within the region showing country-based differences, and hence more likely to form clonal lineages. MRSA is endemic in this region, and the burden and the difficulty in detecting imported strains are increasing. This is also increasing the risk of domestic and global transmission. To counter the threat associated with the high incidence of MRSA carriage and infections, systematic surveillance of both hospital and community isolates is required, along with appropriate measures designed to limit their spread. Additionally, antibiotic stewardship is needed to contain the further development of the observed resistance and to help in preserving antibiotics as precious therapeutic resources. It is critical for countries in this region to establish both national and international initiatives to develop better measurements designed to limit and control the spread of infections. Finally, more sequence-based studies are needed to better understand the pathogenicity and epidemiology of these important pathogens.

The usefulness of whole genome sequencing in the management of Staphylococcus aureus infections.

Staphylococcus aureus remains a leading cause of hospital-acquired and community-associated infection worldwide. The burden of disease is exacerbated by the emergence of virulent strains with reduced susceptibility to commonly used antibiotics and their dissemination in healthcare settings and in the community. Whole genome sequencing (WGS) has the potential to revolutionize our understanding and management of S. aureus infection. As a research tool, WGS has provided insights into the origins of antibiotic-resistant strains, the genetic basis of virulence, the emergence and spread of lineages, and the population structure of S. aureus. As a frontline tool, WGS offers the prospect of a method that could be used to predict resistance, assess virulence, and type isolates at the highest possible resolution. The results generated could be used to guide clinical management and infection control practice. Studies using bench-top sequencing machines have already demonstrated the feasibility of such approaches. Infection control management is compromised by our incomplete understanding of transmission, which in turn reflects the suboptimal resolution offered by conventional typing methods. As the costs of sequencing begin to approach those of conventional methods, high-resolution typing with WGS could realistically be implemented for hospital infection control, as well as for local and national surveillance practice. Translation into routine practice will require the development of a knowledge base, reliable automated bioinformatic tools, the capacity to store, exchange and interrogate large volumes of genomic data, and an acceptance of WGS by clinicians, infection control specialists, and laboratory staff.

Trends and characteristics of culture-confirmed Staphylococcus aureus infections in a large U.S. integrated health care organization.

Infections due to Staphylococcus aureus present a significant health problem in the United States. Between 1990 and 2005, there was a dramatic increase in community-associated methicillin-resistant S. aureus (MRSA), but recent reports suggest that MRSA may be declining. We retrospectively identified S. aureus isolates (n = 133,450) that were obtained from patients in a large integrated health plan between 1 January 1998 and 31 December 2009. Trends over time in MRSA were analyzed, and demographic risk factors for MRSA versus methicillin-susceptible S. aureus (MSSA) were identified. The percentage of S. aureus isolates that were MRSA increased from 9% to 20% between 1998 and 2001 and from 25% to 49% between 2002 and 2005 and decreased from 49% to 43% between 2006 and 2009. The increase in MRSA was seen in blood and in other bacteriological specimens and occurred in all age and race/ethnicity groups, though it was most pronounced in persons aged 18 to <50 years and African-Americans. Hospital onset infections were the most likely to be MRSA (odds ratio [OR], 1.58; confidence interval [CI], 1.46 to 1.70, compared to community-associated cases), but the largest increase in MRSA was in community-associated infections. Isolates from African-Americans (OR, 1.73; CI, 1.64 to 1.82) and Hispanics (OR, 1.11; CI, 1.06 to 1.16) were more likely to be MRSA than those from whites. After substantial increases between 1998 and 2005 in the proportion of S. aureus isolates that were MRSA, the proportion decreased between 2006 and 2009. Hospital onset S. aureus infections are disproportionately MRSA, as are those among African-Americans.

Evaluation of adding a second marker to overcome Staphylococcus aureus spa typing homoplasies.

The utility of sequencing a second highly variable locus in addition to the spa gene (e.g., double-locus sequence typing [DLST]) was investigated to overcome limitations of a Staphylococcus aureus single-locus typing method. Although adding a second locus seemed to increase discriminatory power, it was not sufficient to definitively infer evolutionary relationships within a single multilocus sequence type (ST-5).

Bacterial load of fresh vegetables and their resistance to the currently used antibiotics in Saudi Arabia.

This study was carried out to describe the bacterial load and the occurrence of some disease-causing enteric bacteria on raw vegetables sold in Saudi markets. The study further aimed to analyze antibiotic resistance rates, production of extended-spectrum beta lactamase, and plasmid carriage among bacterial population of raw vegetables. Results revealed that none of them contained Bacillus cereus, Salmonella, and Escherichia coli O157:H7. However, Staphylococcus aureus and Shigella were detected in 11.8% and 4.4% of the samples, respectively. The bacterial loads ranged from 3 to 8 log(10) CFUg(-1) for aerobic bacteria and 1 to 4 log(10) CFUg(-1) for coliforms as well as Enterobacteriaceae. The isolates exhibited resistance in decreasing order for ampicillin (76.5%), cephalothin (69.5%), trimethoprime-sulfamethoxazole (36.7%), aminoglycosides (21.9%), tetracycline (17.2%), fluoroquinolones (17.2%), amoxycillin-clavulanic acid (13.3%), and chloramphenicol (7.8%). Maximum resistance to extended-spectrum beta-lactam antibiotics occurred in 14.8% of isolates and the production of extended-spectrum beta-lactamase was achieved by 2.3% of isolates. Multiple resistances to four or more antimicrobial agents along with plasmid with varied sizes were documented. These investigations indicate the occurrence of antibiotic resistance and plasmid carriage among bacterial isolates populating raw vegetables.

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing.

BACKGROUND: Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. RESULTS: Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. CONCLUSIONS: This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.

Development of a modified DNA extraction method for pulsed-field gel electrophoresis analysis of Staphylococcus aureus and enterococci without using lysostaphin.

A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of Staphylococcus aureus and enterococci to reduce the cost of using lysostaphin. This protocol reduces the expenses of PFGE typing of S. aureus and enterococci as it removes the use of lysostaphin during the spheroplast formation from these bacteria.

Staphylococcus aureus nosocomial infections: the role of a rapid and low-cost characterization for the establishment of a surveillance system.

Continuous surveillance on resistance patterns and characterization of Staphylococcus aureus represent simple and low-cost techniques to understand and evaluate the effectiveness of infection control and antimicrobial prescribing measures. In this study we analyzed the antibiotic susceptibility and trends for S. aureus strains collected from bacteraemia cases in a five year period. Between 2004 and 2008 we noted a progressive decrease in the number of S. aureus isolates compared to all pathogens from clinical specimens and S. aureus bloodstream infections (BSI) reflected a similar trend. In particular we analyzed 185 isolates from blood cultures: 89 isolates were MSSA and 96 isolates were MRSA. Molecular SCCmec typing of these strains showed an absolute prevalence of types I and II, whereas five spa types from 96 isolates were obtained. Resistance pattern analysis allowed us to place MRSA strains into 12 antibiotypes and the major antibiotype was resistant to penicillin, gentamicin, erythromycin, clindamycin and ciprofloxacin. The predominant antibiotype among the MSSA isolates was resistant only to penicillin. In addition, 19.1% of MSSA are susceptible to all antibiotics tested. We also found a close association between antibiotyping 1 and genotyping t002/SCCmecI of MRSA strains, suggesting a nosocomial scenario dominated by a few particular clones.

De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer.

Novel high-throughput DNA sequencing technologies allow researchers to characterize a bacterial genome during a single experiment and at a moderate cost. However, the increase in sequencing throughput that is allowed by using such platforms is obtained at the expense of individual sequence read length, which must be assembled into longer contigs to be exploitable. This study focuses on the Illumina sequencing platform that produces millions of very short sequences that are 35 bases in length. We propose a de novo assembler software that is dedicated to process such data. Based on a classical overlap graph representation and on the detection of potentially spurious reads, our software generates a set of accurate contigs of several kilobases that cover most of the bacterial genome. The assembly results were validated by comparing data sets that were obtained experimentally for Staphylococcus aureus strain MW2 and Helicobacter acinonychis strain Sheeba with that of their published genomes acquired by conventional sequencing of 1.5- to 3.0-kb fragments. We also provide indications that the broad coverage achieved by high-throughput sequencing might allow for the detection of clonal polymorphisms in the set of DNA molecules being sequenced.

External quality assessment of molecular typing of Staphylococcus aureus isolates by a network of laboratories.

A network of laboratories designated Centres for Molecular Diagnosis was funded in 2000 by Belgian National Health Insurance to provide clinically relevant molecular diagnostic tests. These included typing of nosocomial pathogens as a service to local hospital infection control programs. Two external quality assessment (EQA) surveys were performed in 2001 and 2003 to evaluate the proficiencies of the laboratories at Staphylococcus aureus typing. EQA panels included S. aureus isolates with either indistinguishable, clonally related, or unrelated pulsed-field gel electrophoresis (PFGE) patterns. A hypothetical hospital outbreak problem was also submitted for analysis. Typeability, reproducibility, discrimination (D) index, and epidemiological concordance were evaluated. Ten centers participated in each survey. Seven centers performed PFGE analysis, while others used repetitive-element or randomly amplified polymorphic DNA PCR, amplified fragment length polymorphism, or spa typing. Full typeability (100%) was achieved by all centers, and all but one showed 100% reproducibility. Discrimination was appropriate (D index, >or=96%) for centers performing PFGE analysis but not for all those using other methods (D index range, 72% to 97%). Correct answers to the epidemiological questions were provided by 7/10 and 10/10 centers in 2001 and 2003, respectively. Individual feedback of results was provided to each center together with specific technical recommendations for improving performance. Our findings indicate that surveys of lab proficiency are useful for validation and optimization of molecular typing services to local hospital infection control programs.

Rapid cost-effective subtyping of meticillin-resistant Staphylococcus aureus by denaturing HPLC.

The importance of meticillin-resistant Staphylococcus aureus (MRSA) in hospital-acquired infection is widely acknowledged. The UK government has stated that MRSA bloodstream infection rates will have to be halved by 2008. Such radical improvements will require advances on several fronts. Screening for MRSA in high-risk patients on arrival at hospital allows isolation of carriers and reduces transmission to staff and other patients. Concurrent subtyping of MRSA could also inform outbreak investigations and long-term epidemiological studies. The variability within the staphylococcal protein A, or spaA, gene-repeat region can be used as a marker of short- and long-term genetic variation. A novel application is described of denaturing HPLC (DHPLC) for rapid, inexpensive characterization of spaA gene amplification products, without the need for DNA sequence determination. The method allowed rapid and precise sizing of spaA gene-repeat regions from 99 S. aureus strains and was combined with heteroduplex analysis, using reference PCR products, to indicate the spa type of the test isolate. The method allowed subtyping of strains in less than 5 h from receipt of a primary isolation plate. When applied to an outbreak that occurred during this study, the authors were able to demonstrate relatedness of the isolates more than 5 days before results were received from a reference laboratory. If combined with direct amplification from swabs, DHPLC analysis of spaA gene variation could prove extremely valuable in outbreak investigation and MRSA surveillance.

Use of strain typing data to estimate bacterial transmission rates in healthcare settings.

OBJECTIVE: To create an affordable and accurate method for continuously monitoring bacterial transmission rates in healthcare settings. DESIGN: We present a discrete simulation model that relies on the relationship between in-hospital transmission rates and strain diversity. We also present a proof of concept application of this model to a prospective molecular epidemiology data set to estimate transmission rates for Pseudomonas aeruginosa and Staphylococcus aureus. SETTING: Inpatient units of an academic referral center. PATIENTS: All inpatients with nosocomial infections. INTERVENTION: Mathematical model to estimate transmission rates. RESULTS: Maximum likelihood estimates for transmission rates of these two species on different hospital units ranged from 0 to 0.36 transmission event per colonized patient per day. CONCLUSIONS: This approach is feasible, although estimates of transmission rates based solely on strain typed clinical cultures may be too imprecise for routine use in infection control. A modest level of surveillance sampling substantially improves the estimation accuracy.

Costs and outcomes among hemodialysis-dependent patients with methicillin-resistant or methicillin-susceptible Staphylococcus aureus bacteremia.

OBJECTIVE: Comorbid conditions have complicated previous analyses of the consequences of methicillin resistance for costs and outcomes of Staphylococcus aureus bacteremia. We compared costs and outcomes of methicillin resistance in patients with S. aureus bacteremia and a single chronic condition. DESIGN, SETTING, AND PATIENTS: We conducted a prospective cohort study of hemodialysis-dependent patients with end-stage renal disease and S. aureus bacteremia hospitalized between July 1996 and August 2001. We used propensity scores to reduce bias when comparing patients with methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus bacteremia. Outcome measures were resource use, direct medical costs, and clinical outcomes at 12 weeks after initial hospitalization. RESULTS: Fifty-four patients (37.8%) had MRSA and 89 patients (62.2%) had MSSA. Compared with patients with MSSA bacteremia, patients with MRSA bacteremia were more likely to have acquired the infection while hospitalized for another condition (27.8% vs 12.4%; P = .02). To attribute all inpatient costs to S. aureus bacteremia, we limited the analysis to 105 patients admitted for suspected S. aureus bacteremia from a community setting. Adjusted costs were higher for MRSA bacteremia for the initial hospitalization (21,251 dollars vs 13,978 dollars; P = .012) and after 12 weeks (25,518 dollars vs 17,354 dollars; P = .015). At 12 weeks, patients with MRSA bacteremia were more likely to die (adjusted odds ratio, 5.4; 95% confidence interval, 1.5 to 18.7) than were patients with MSSA bacteremia. CONCLUSIONS: Community-dwelling, hemodialysis-dependent patients hospitalized with MRSA bacteremia face a higher mortality risk, longer hospital stays, and higher inpatient costs than do patients with MSSA bacteremia.

[Evaluation of three methods for genotyping of nosocomial methicillin resistant Staphylococcus aureus isolates]. / Nozokomiyal metisiline dirençli Staphylococcus aureus izolatlarinin genotiplendirilmesinde üç ayri yöntemin incelenmesi.

Plasmid profile analysis (PPA), random amplification of polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE) are the three most valuable epidemiological tools for genotyping of methicillin resistant Staphylococcus aureus (MRSA) strains. Aim of this study was to evaluate these three methods in respect to their cost, reproducibility, and discriminatory power. Eighty one nosocomial MRSA isolates with unknown genetic and epidemiological relatedness from Training Hospital of Gulhane Military Medical School, were genotyped by PPA, RAPD, and PFGE methods. All isolates (100%) were typed by RAPD and PFGE, however, eight (9.9%) isolates could not be typed by PPA since they lacked plasmid DNA. Reproducibilities of all the three methods were found to be 100 percent. Discriminatory powers of PPA, RAPD and PFGE methods were calculated as 48.6%, 61.1% and 80.1%, respectively. In conclusion, out of the three methods tested, PFGE allowed the most effective discrimination of MRSA strains. However, PFGE was more time consuming and technically demanding, and required use of specialized and expensive equipment. Although PPA and RAPD were less discriminatory than PFGE, these methods were technically simple, rapid and cheaper. When PPA and RAPD were used in combination, they had equal discriminatory power to PFGE. Thus, it should be emphasized that PPA and RAPD methods could be preferred for initial screening purposes while PFGE should be used as a confirmatory test in genotyping of MRSA isolates.

Molecular typing of methicillin-resistant Staphylococcus aureus: can PCR replace pulsed-field gel electrophoresis?

Pulsed-field gel electrophoresis (PFGE) is considered the "gold standard" for molecular typing of methicillin-resistant Staphylococcus aureus (MRSA). However, the method is time-consuming and expensive, and its discriminatory power may not be necessary in outbreak situations. We used a rapid multiplex PCR-based method with published primers and compared the results with those obtained by PFGE. A total of 75 clinical isolates were typed: 59 strains originated from our prospectively collected clinical strains and were epidemiologically unrelated; 16 strains came from an outbreak that was epidemiologically well defined in time and space. A primer mix of the spa gene, the coa gene, and the hypervariable region adjacent to mecA gene was used for multiplex PCR. Both PFGE and PCR clustered the 75 strains into 41 different genotypes. Concordance of the results was 100% for strains originating from the outbreak. Overall, both methods produced concordant results in 72% of cases. A total of 16% were clustered together by PFGE, but not by PCR and 12% were clustered together by PCR but not by PFGE, respectively. The turnaround time was only 8 h for PCR but 5 days for PFGE. This PCR-based method is excellent for rapid and inexpensive typing of MRSA in an outbreak setting, but the discriminatory power and reproducibility are still insufficient to replace PFGE in longitudinal studies in the endemic setting.