Comparison of Two Multilocus Variable-Number Tandem-Repeat Methods and Pulsed-Field Gel Electrophoresis for Differentiating Highly Clonal Methicillin-Resistant Staphylococcus aureus Isolates

In the United Kingdom, EMRSA-15 and EMRSA-16 account for the majority (∼90%) of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the standard typing technique, pulsed-field gel electrophoresis (PFGE), is laborious and insufficient for discriminating between closely related subtypes of EMRSA-15 and -16. The objective of the present study was to compare the usefulness of multilocus variable-number tandem-repeat fingerprinting (MLVF) and multilocus variable-number tandem-repeat analysis (MLVA) with PFGE for subtyping these highly clonal MRSA lineages. A panel of 85 MRSA isolates (41 EMRSA-15, 20 EMRSA-16, and 24 MRSA isolates with diverse PFGE patterns) was investigated. In addition, a further 29 EMRSA-15s with identical PFGE patterns from two geographically linked but epidemiologically distinct outbreaks and several sporadic cases were analyzed. PFGE, MLVF, and MLVA resolved 66 (Simpson''s index of diversity [SID] = 0.984), 51 (SID = 0.95), and 42 (SID = 0.881) types, respectively, among the 85 MRSA isolates. MLVF was more discriminatory than MLVA for EMRSA-15 and -16 strains, but both methods had comparable discriminatory powers for distinguishing isolates in the group containing diverse PFGE types. MLVF was comparable to PFGE for resolving the EMRSA-15s but had a lower discriminatory power for the EMRSA-16s. MLVF and MLVA resolved the 29 isolates with identical PFGE patterns into seven and six subtypes, respectively. Importantly, both assays indicated that the two geographically related outbreaks were caused by distinct subtypes of EMRSA-15. Taken together, the data suggest that both methods are suitable for identifying and tracking specific subtypes of otherwise-indistinguishable MRSA. However, due to its greater discriminatory power, MLVF would be the most suitable alternative to PFGE for hospital outbreak investigations.Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen causing considerable morbidity and mortality worldwide. In Scotland and the United Kingdom in general, the incidence of MRSA is high (∼35%) with two epidemic clones, EMRSA-15 (ST22) and EMRSA-16 (ST36/ST30), accounting for 70 and 20%, respectively, of isolates referred to the Scottish MRSA Reference Laboratory (SMRSARL). Molecular typing of clinical isolates is important to inform decisions regarding effective control measures. For over a decade, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA has been used in United Kingdom reference laboratories for outbreak investigations and epidemiological surveillance, owing to its high discriminatory power and validated interpretation scheme (18). However, it is laborious and time-consuming, with poor interlaboratory comparability, and has no common international nomenclature. Furthermore, ca. 50% of EMRSA-15 strains and 35% of EMRSA-16 strains are indistinguishable by PFGE (4). Accordingly, the method is unsuitable for the tracing of many epidemics caused by EMRSA-15 and EMRSA-16 strains.Various techniques have been developed to address some of the limitations of PFGE, including spa typing and the variable-number tandem-repeat (VNTR)-based methods, multilocus VNTR fingerprinting (MLVF) (15) and multilocus VNTR analysis (MLVA) (6, 13, 16). spa typing involves DNA sequencing of the polymorphic VNTR in the 3′ coding region of the S. aureus-specific staphylococcal protein A (spa) gene (7). The method is more rapid and less laborious than PFGE, and the output is a digital profile, which is easily comparable between laboratories (1). However, it is less discriminatory than PFGE (10, 17) and is unsuitable for investigating the transmission of MRSA in hospitals dominated by EMRSA-15 and EMRSA-16 (8).MLVF analyzes the variation in the number of tandem repeats in seven genes (clfA, clfB, sdrC, sdrD, sdrE, spa, and sspA) by multiplex PCR and has been reported to be highly discriminatory and reproducible (9, 10, 15, 19). Previously, Tenover et al. (19) and Moser et al. (11) demonstrated that MLVF can distinguish between strains with identical PFGE patterns.MLVA differs from MLVF in that the number of repeats at each locus is determined to produce a numerical profile that can be incorporated into electronic databases and easily shared between laboratories. Although several MLVA schemes have been described, which differ in the loci and PCR protocol used, the MLVA described by Schouls et al. (16) benefits from automated fragment sizing on a DNA sequencer.To date, the effectiveness of MLVF and MLVA for tracing hospital outbreaks has not been compared. The aim of the present study was to investigate the usefulness of MLVF and MLVA compared to PFGE for subtyping highly clonal EMRSA-15s (ST22) and EMRSA-16S (ST36/ST30) and for tracing hospital outbreaks of infection.     

Typing of Human Campylobacter jejuni Isolates in Finland by Pulsed-Field Gel Electrophoresis

A total of 69 pulsed-field gel electrophoresis (PFGE) types were identified among 176 Campylobacter jejuni isolates from Finnish patients. In two geographic areas studied, five predominant PFGE types comprised over 40% of the isolates. One-third of the isolates had unique PFGE types. In small outbreaks, identical PFGE patterns were demonstrated, indicating a common source of infection.     

Comparison of Restriction Enzymes for Pulsed-Field Gel Electrophoresis Typing of Moraxella catarrhalis

NotI, the most prevalent restriction enzyme used for typing Moraxella catarrhalis, failed to digest genomic DNA from respiratory samples. An improved pulsed-field gel electrophoresis (PFGE) methodology determined SpeI as the best choice for typing this bacterial species, with a good restriction of clinical samples and a good clustering correlation with NotI.     

Typing of Campylobacter jejuni Isolates from Dogs by Use of Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

Campylobacter is a major cause of human gastroenteritis worldwide. Risk of Campylobacter infection in humans has been associated with many sources, including dogs. This study aimed to investigate whether C. jejuni carried by dogs could potentially be a zoonotic risk for humans and if there were common sources of C. jejuni infection for both humans and dogs. Multilocus sequence typing (MLST) together with macrorestriction analysis of genomic DNA using SmaI and pulsed-field gel electrophoresis (PFGE) were both used to analyze 33 C. jejuni isolates obtained from various dog populations, including those visiting veterinary practices and from different types of kennels. MLST data suggested that there was a large amount of genetic diversity between dog isolates and that the majority of sequence types found in isolates from these dogs were the same as those found in isolates from humans. The main exception was ST-2772, which was isolated from four samples and could not be assigned to a clonal complex. The most commonly identified clonal complex was ST-45 (11 isolates), followed by ST-21 (4 isolates), ST-508 (4 isolates), and ST-403 (3 isolates). The profiles obtained by macrorestriction PFGE were largely in concordance with the MLST results, with a similar amount of genetic diversity found. The diversity of sequence types found within dogs suggests they are exposed to various sources of C. jejuni infection. The similarity of these sequence types to C. jejuni isolates from humans suggests there may be common sources of infection for both dogs and humans. Although only a small number of household dogs may carry C. jejuni, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel dog populations, where the prevalence may be higher.Campylobacter species are among the most commonly reported bacterial cause of human gastroenteritis (2, 5, 10, 44). The majority of such infections are caused by C. jejuni and C. coli. There is some evidence of an elevated risk of Campylobacter infection in humans associated with dog or pet ownership (1, 17, 38).Multilocus sequence typing (MLST) is a tool used to investigate relationships between Campylobacter spp., particularly C. jejuni. It is useful for investigating possible reservoir hosts and host associations and also for studying the molecular epidemiology of the disease (11, 28). The C. jejuni clonal complexes ST-21 and ST-45 and others, such as ST-61, are often isolated from human cases of campylobacteriosis (12, 31). The complexes ST-21 and ST-45 are commonly found in chickens, cattle, water, and wildlife, and ST-61 has been particularly associated with cattle (7, 18, 22, 36).Some other C. jejuni sequence types isolated from humans have not yet been associated with cattle and poultry, and the sources of these need further investigation (31). In contrast, some sequence types are strongly associated with certain animal hosts (6) but are not usually isolated from humans (12, 13, 15, 31).Whether or not dogs are a possible source of C. jejuni infection for humans is not fully understood. Macrorestriction pulsed-field gel electrophoresis (PFGE) has been used in studies to compare C. jejuni isolates found in both dogs and humans (29), and in some cases the dog and the owner did share an identical strain (8). There is currently little MLST data available for Campylobacter spp. isolated from dogs (16; http://pubmlst.org/campylobacter [accessed 5 February 2009]), although ST-45 infection in humans has also been significantly associated with contact with pet cats and dogs (22).The aims of this study were to examine 33 C. jejuni isolates obtained from dogs from various populations using MLST in order to determine whether strains of C. jejuni carried by dogs appear to be different from those found in humans and other species and to determine possible sources of canine infection. PFGE with SmaI digestion was also performed with the isolates in order to investigate the epidemiology of the disease within the different dog populations.     

Comparison of an Automated Repetitive-Sequence-Based PCR Microbial Typing System with Pulsed-Field Gel Electrophoresis for Molecular Typing of Vancomycin-Resistant Enterococcus faecium

Vancomycin-resistant Enterococcus faecium (VRE) has become an important health care-associated pathogen because of its rapid spread, limited therapeutic options, and possible transfer of vancomycin resistance to more-virulent pathogens. In this study, we compared the ability to detect clonal relationships among VRE isolates by an automated repetitive-sequence-based PCR (Rep-PCR) system (DiversiLab system) to pulsed-field gel electrophoresis (PFGE), the reference method for molecular typing of VRE. Two sets of VRE isolates evaluated in this study were collected by active microbial surveillance at a large teaching hospital in Taiwan during 2008. The first set included 90 isolates randomly selected from the surveillance cohort. The first set consisted of 34 pulsotypes and 10 Rep-PCR types. There was good correlation between the two methods (P < 0.001). The second set included 68 VRE isolates collected from eight clusters of colonization. A dominant clone was detected in five out of eight clusters by both methods. Two clusters were characterized by Rep-PCR as being caused by a dominant clone, whereas PFGE showed polyclonal origins. One cluster was shown to be polyclonal by both methods. A single Rep-PCR clone type was detected among 12 of 14 vancomycin-intermediate enterococci, whereas PFGE detected six pulsotypes. In conclusion, the Rep-PCR method correlated well with PFGE typing but was less discriminative than PFGE in defining clonal relationships. The ease of use and more rapid turnaround time of Rep-PCR compared to PFGE offers a rapid screening method to detect outbreaks of VRE and more rapidly implement control measures. PFGE remains the preferred method to confirm clonal spread.Vancomycin-resistant Enterococcus faecium (VRE) was first isolated in England in 1986 (26). It has become an important health care-associated pathogen because of its rapid spread, significant attributable mortality, limited options for therapy, and the possible transfer of vancomycin resistance to more-virulent pathogens (3, 4, 14). VRE organisms are now commonly isolated in hospitals around the world. Molecular typing methods are needed to distinguish prevalent strains from epidemic strains. Pulsed-field gel electrophoresis (PFGE) is the accepted reference method for molecular typing of VRE chromosomes because of its high discriminative power and established interpretative guidelines (25, 29). PFGE is labor-intensive and skill dependent, and the turnaround time is 2 to 3 days (7, 27). VRE isolates collected during outbreak investigations at our hospital during the past 2 years consisted of a wide variety of PFGE types. Polyclonal outbreaks have also been reported by other investigators (8, 12). It is not clear whether this represents multiple introductions of different strains or microevolutionary changes in native VRE or whether PFGE is overly discriminatory.Repetitive-sequence-based PCR (Rep-PCR) methods are rapid typing procedures that amplify the regions between the noncoding repetitive sequences in bacterial genomes (28). Recently, Healy et al. described an automated Rep-PCR technology and its performance characteristics (10). Rep-PCR has been used in VRE outbreak investigations (20, 23), but we are unaware of any comparative field tests of Rep-PCR and PFGE. This study was designed to compare the ability of the two methods to distinguish clonal relationships among a panel of epidemiologically well-defined isolates of VRE. We also evaluated the association of specific drug resistance patterns with selected genotypes.     

Molecular Typing of Penicillium marneffei Isolates from Thailand by NotI Macrorestriction and Pulsed-Field Gel Electrophoresis

Penicillium marneffei is recognized as one of the most frequently detected opportunistic pathogens of AIDS patients in northern Thailand. We undertook a genomic epidemiology study of 64 P. marneffei isolates collected from immunosuppressed patients by pulsed-field gel electrophoresis (PFGE) with restriction enzyme NotI. Among the 69 isolates fingerprinted by PFGE, 17 were compared by HaeIII restriction endonuclease typing. The PFGE method demonstrated a higher degree of discriminatory power than restriction endonuclease typing with HaeII. Moreover, an impressive diversity of P. marneffei isolates was observed, as there were 54 distinct macrorestriction profiles among the 69 isolates of P. marneffei. These profiles were grouped into two large clusters by computer-assisted similarity analysis: macrorestriction pattern I (MPI) and MPII, with nine subprofiles (MPIa to MPIf and MPIIa to MPIIc). We observed no significant correlation between the macrorestriction patterns of the P. marneffei isolates and geographical region or specimen source. It is interesting that all isolates obtained before 1995 were MPI, and we found an increase in the incidence of infections with MPII isolates after 1995. We conclude that PFGE is a highly discriminatory typing method and is well suited for computer-assisted analysis. Together, PFGE and NotI macrorestriction allow reliable identification and epidemiological characterization of isolates as well as generate a manageable database that is convenient for expansion with information on additional P. marneffei isolates.     

Comparing Pulsed-Field Gel Electrophoresis with Multilocus Sequence Typing,spa Typing,Staphylococcal Cassette Chromosome mec (SCCmec) Typing,and PCR for Panton-Valentine Leukocidin,arcA, and opp3 in Methicillin-Resistant Staphylococcus aureus Isolates at a U.S. Medical Center

Methicillin-resistant Staphylococcus aureus (MRSA) has become a common cause of skin infections and invasive infections in community dwellers in the United States since the late 1990s. Isolates characterized as USA300 by pulsed-field gel electrophoresis (PFGE) are the predominant strain type in these infections. USA100 and USA500 strains commonly cause health care-associated infections. We compared PFGE with a number of other methods of genotyping in a sample of 149 clinical MRSA isolates from the University of Chicago Medical Center. The 5 USA500 isolates yielded 3 spa types and 2 multilocus sequence types (MLSTs). Among the 24 USA100 isolates, 21 (88%) were of spa type t002, 19 (79%) were of ST5, 2 carried arcA and opp3, and 1 was Panton-Valentine leukocidin positive (PVL⁺). Among the 102 USA300 isolates, 96 (94%) were of ST8 and 94 (92%) were of spa type t008. The combination of traits that provided the best sensitivity (98%), specificity (97%), positive predictive value (PPV) (99%), and negative predictive value (NPV) (95%) for identifying USA300 isolates were the presence of the arcA gene and the presence of the PVL genes (area under the curve, 0.980; 95% confidence interval [CI], 0.955 to 1.0). PFGE did not delineate a homogeneous group of MRSA genetic backgrounds, as documented for other typing methods, particularly for USA500 and USA100 pulsotypes. Documenting the presence of arcA and PVL genes by PCR was an efficient and accurate means of identifying USA300 in a collection of MRSA isolates in which USA300 is common. None of the tested genotyping methods provided an accurate means of identifying the next most common PFGE-based backgrounds, USA100 and USA500.     

Comparison of Fingerprinting Methods for Typing Methicillin-Resistant Staphylococcus aureus Sequence Type 398

This study evaluates the multiple-locus variable-number tandem-repeat assay (MLVA) and pulsed-field gel electrophoresis (PFGE) when using restriction enzymes BstZI, SacII, and ApaI to fingerprint a diverse collection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) sequence type 398 (ST398) isolates. These isolates had been characterized previously by multilocus sequence typing, spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Typeability and discriminatory power were analyzed, and the concordance between the various methods was determined. All MRSA ST398 isolates were typeable by the MLVA and PFGE using BstZI, SacII, and ApaI. With each method, the MRSA ST398 isolates formed a separate group from the two non-ST398 MRSA strains. PFGE, performed with any of the three restriction enzymes, had the most discriminatory power, followed by MLVA, spa typing, and SCCmec typing. The MLVA showed the highest concordance with PFGE using ApaI and spa typing. As further expressed by the Wallace coefficient, the MLVA type was poorly predicted by spa typing, whereas the spa type was well predicted by MLVA. PFGE, using a combination of all three restriction enzymes, had the highest concordance with the MLVA but had a low probability of being predicted by MLVA. PFGE, using a combination of all three restriction enzymes, was able to predict SCCmec type and MLVA type completely and had a high probability of predicting spa type. Both the MLVA and PFGE could be used to discriminate among the MRSA ST398 isolates. Although the MLVA is a faster technique, PFGE had more discriminatory power than the MLVA, especially when a combination of restriction enzymes was used.Infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) have been a problem in hospitals and nursing homes for many decades. These MRSA isolates are therefore called health care-associated MRSA (HA-MRSA) isolates (1). Since the early 1990s, MRSA has emerged in healthy persons without risk factors for MRSA infections. These isolates are the so-called community-associated MRSA (CA-MRSA) isolates (20). In the last few years, MRSA has been isolated from livestock animals (pigs in particular) and pig farmers (5, 6, 30). These MRSA strains are called animal-associated MRSA (AA-MRSA) strains. It seems that (livestock) animals form a new, separate reservoir. These AA-MRSA strains all appear to belong to the new clonal complex 398 (CC398), with sequence type 398 (ST398) as the basic type, as determined by multilocus sequence typing (MLST) (29). MRSA ST398 has already been isolated in Europe, Asia, and North America (32). Considering the worldwide spread of MRSA, epidemiological questions arise about its transmission within farms, among farms, and from farms to the population. Fast and inexpensive typing methods with good discriminatory power are necessary to conduct large-scale epidemiological studies.Traditionally, human MRSA isolates have been typed by pulsed-field gel electrophoresis (PFGE), using SmaI as the restriction enzyme (19). The advantages of using PFGE are good discriminatory power and good reproducibility at the interlaboratory level when standardized protocols are used. However, AA-MRSA is not typeable by this method, as the activity of SmaI is blocked due to methylation of the restriction site (2).More recently, methods based on DNA sequencing, such as MLST and spa typing, are increasingly being used to discriminate among different MRSA strains. Given their excellent interlaboratory reproducibility, online databases have been made to collate and harmonize data from various geographic regions. The drawback of MLST, which measures sequence variation at seven housekeeping loci, is its limited use with epidemiological studies due to its weak discriminatory power, time-consuming protocols, and high costs. spa typing, based on the variation in repeats present in the X-region of staphylococcal protein A, has a discriminatory power that lies between those of PFGE and MLST. Within ST398, several spa types have been distinguished, although the number of spa types seems rather limited in most countries.One promising method is the multiple-locus variable-number tandem-repeat assay (MLVA), a PCR-based method, based on the analysis of the number of repeats in the variable-number tandem-repeat regions of various individual genes. This method has proven to be useful for typing both Staphylococcus aureus and clinical MRSA isolates with good reproducibility and good discriminatory power. Because the MLVA is also simple, inexpensive, and easy to interpret, it is useful as a typing method for large-scale epidemiological studies (10, 11, 12, 15, 16, 17, 23, 24, 27).This study aimed to investigate various methods for typing MRSA ST398 isolates. An MLVA, consisting of a selection of primers from three existing MLVA systems, was tested with a collection of MRSA ST398 isolates. In addition, PFGE with restriction enzymes other than SmaI was performed with this set of isolates. These isolates had been previously characterized by MLST, spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Typeability and discriminatory power were analyzed for all methods, and the concordance among the different methods was determined.     

Comparison of Multiple-Locus Variable-Number Tandem-Repeat Analysis with Pulsed-Field Gel Electrophoresis Typing of Acinetobacter baumannii in China

A panel of seven variable-number tandem-repeat (VNTR) markers was selected for Acinetobacter baumannii typing analysis (MLVA-7). Compared with pulsed-field gel electrophoresis (PFGE), MLVA-7 provided greater discrimination. We modified the criteria for MLVA complex assignments proposed previously, and a remarkable congruence between MLVA-7- and PFGE-based strain clustering was observed.     

Typing of Salmonella enterica Serotype Paratyphi C Isolates from Various Countries by Plasmid Profiles and Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) of 61 Salmonella enterica serotype Paratyphi C isolates from six countries gave five distinct clusters. Twenty-four isolates from five countries were susceptible to 10 antimicrobials tested and gave similar restriction endonuclease digest patterns of the 38-MDa plasmid. In contrast, plasmid and PFGE profiles of 37 multidrug-resistant isolates from Zaire were different from those from other countries.     

Comparison of Randomly Amplified Polymorphic DNA Analysis and Pulsed-Field Gel Electrophoresis for Typing of Moraxella catarrhalis Strains

Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for the analysis of 13 Moraxella catarrhalis isolates, 11 successive strains isolated from sputa of five children and 2 isolates obtained the same day from twins, were compared. RAPD and PFGE both yielded nine types from the 13 isolates, showing a chronic colonization with one strain in three patients and a successive colonization with different strains in two patients. The promising results obtained with RAPD should be confirmed with a larger number of strains, but RAPD seems as suitable as PFGE for the typing of M. catarrhalis.     

Analysis of Legionella pneumophila Serogroup 1 Isolates by Pulsed-Field Gel Electrophoresis

At a hospital in Halifax, Nova Scotia, Canada, three strains of Legionella pneumophila were detectable based on plasmid content, while the isolates collected at another hospital in Halifax had no plasmids. Genomic DNA was digested with BssHII, SalI, and SpeI and subjected to pulsed-field gel electrophoresis (PFGE). We found no relationship between plasmid profile and PFGE pattern.     

Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Clinical Isolates Obtained from the Rikers Island Jail System from 2009 to 2013

Inmates of Rikers Island jail potentially introduce Staphylococcus aureus into New York State prisons upon transfer. In this study, methicillin-resistant Staphylococcus aureus isolates (n = 452), collected from infected inmates (2009 to 2013), were characterized. spa type t008 was the predominant clone identified, accounting for 82.3% of the isolates, with no evidence of mupirocin or chlorhexidine resistance.     

Epidemiological Analysis of Salmonella enteritidis Isolates from Humans and Broiler Chickens in Thailand by Phage Typing and Pulsed-Field Gel Electrophoresis

To determine the phage types (PT) of Salmonella enteritidis found in Thailand and to clarify the potential for human infection by S. enteritidis in broiler chicken meat, human and poultry isolates taken from Thailand between 1990 and 1997 were phage typed and analyzed by pulsed-field gel electrophoresis (PFGE). Ten different PT were found among the 302 isolates phage typed, with PT 4 being the most frequent in human (73.9%) and poultry (76.2%) isolates, followed by PT 1 (8.0%), 8 (3.6%), and 7a (2.2%) in human isolates and by PT 7a (4.9%), 1 (3.7%), and 12 (2.4%) in poultry isolates. Of the 53 isolates analyzed by PFGE, 45 showed an indistinguishable pattern (pattern A) by BlnI-digested PFGE and the other 8 isolates showed a very similar pattern that differed by only a few bands. These results indicate the spread of a genetically identical clone of S. enteritidis in humans and poultry in Thailand.     

Prospective Genotyping of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Isolates by Use of a Novel,Highly Discriminatory Binary Typing System

In settings of high methicillin-resistant Staphylococcus aureus (MRSA) prevalence, detection of nosocomial transmission events can be difficult without strain typing. Prospective typing of all MRSA isolates could potentially identify transmission in a timely fashion, making infection control responses to outbreaks more effective. We describe the development and evaluation of a novel 19-target binary typing system for MRSA using the multiplex-PCR/reverse line blot hybridization platform. Pulse-field gel electrophoresis (PFGE), spa typing, and phage-derived open reading frame (PDORF) typing were performed for comparison. The system was utilized to identify transmission events in three general surgical wards over a 12-month period. Initial MRSA isolates from 273 patients were differentiated into 55 unique binary types. One or more potential contacts colonized with the same MRSA strain were identified in 69 of 87 cases (79%) in which definite or possible nosocomial MRSA acquisition had occurred. The discriminatory power of the typing system was similar to that of PFGE (Simpson''s index of diversity [D] = 0.994, versus 0.987) and higher than that of spa typing (D = 0.926). Strain typing reduced the total number of potential MRSA-colonized source contacts from 859 to 212 and revealed temporal clustering of transmission events. Prospective MRSA typing using this novel binary typing method can rapidly identify nosocomial transmission events, even in high-prevalence settings, which allows timely infection control interventions. The system is rapid, inexpensive, discriminatory, and suitable for routine, high-throughput use in the hospital microbiology laboratory.     

Comparing Whole-Genome Sequencing with Sanger Sequencing for spa Typing of Methicillin-Resistant Staphylococcus aureus

spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and an in-house analysis pipeline determines the spa types. Due to national surveillance, all MRSA isolates are sent to Statens Serum Institut, where the spa type is determined by PCR and Sanger sequencing. The purpose of this study was to evaluate the reliability of the spa types obtained by 150-bp paired-end Illumina WGS. MRSA isolates from new MRSA patients in 2013 (n = 699) in the capital region of Denmark were included. We found a 97% agreement between spa types obtained by the two methods. All isolates achieved a spa type by both methods. Nineteen isolates differed in spa types by the two methods, in most cases due to the lack of 24-bp repeats in the whole-genome-sequenced isolates. These related but incorrect spa types should have no consequence in outbreak investigations, since all epidemiologically linked isolates, regardless of spa type, will be included in the single nucleotide polymorphism (SNP) analysis. This will reveal the close relatedness of the spa types. In conclusion, our data show that WGS is a reliable method to determine the spa type of MRSA.     

Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships.     

Two Distinct Clones of Methicillin-Resistant Staphylococcus aureus (MRSA) with the Same USA300 Pulsed-Field Gel Electrophoresis Profile: a Potential Pitfall for Identification of USA300 Community-Associated MRSA

Analysis of methicillin-resistant Staphylococcus aureus (MRSA) characterized as USA300 by pulsed-field gel electrophoresis identified two distinct clones. One was similar to community-associated USA300 MRSA (ST8-IVa, t008, and Panton-Valentine leukocidin positive). The second (ST8-IVa, t024, and PVL negative) had different molecular characteristics and epidemiology, suggesting independent evolution. We recommend spa typing and/or PCR to discriminate between the two clones.The methicillin-resistant Staphylococcus aureus (MRSA) clone USA300, having multilocus sequence type (MLST) ST8 and staphylococcal protein A (spa) type 008 and carrying staphylococcal cassette chromosome (SCCmec) IVa, has disseminated in the United States, as well as to other parts of the world (16, 26, 27). USA300 carries the luk-PV genes encoding Panton-Valentine leukocidin (PVL), has been identified in a variety of community populations, and has been associated with skin and soft tissue infections (SSTI), as well as more severe infections, such as sepsis, pneumonia, and necrotizing fasciitis (7, 22, 28).The identification of USA300 isolates is primarily based on pulsed-field gel electrophoresis (PFGE) (10). Other genetic markers have also been suggested for identification of USA300 isolates, including (i) the arcA gene of the arginine catabolic mobile element (ACME) (3, 8, 26, 29), (ii) sequencing of the direct repeat unit (dru) region (9), and (iii) different USA300-specific multiplex PCRs targeting luk-PV and a “signature” six-AT-repeat sequence within the conserved hypothetical gene SACOL0058 (2).In Denmark, MRSA isolates have been consecutively typed by PFGE since 1999, with the addition of sequence-based methods, such as MLST and spa typing, on selected isolates since 2001 (6). This process identified some of the first USA300 isolates in Europe but, surprisingly, also identified isolates with USA300 PFGE banding patterns but a different spa type (1, 15, 16). In this study, we investigated the epidemiology and genetic diversity of these isolates and USA300 and USA500 reference strains (20) using PFGE (23), spa typing (11), MLST (5), SCCmec typing (21, 24), dru typing (9), ACME (26), the six-AT signature sequence (2), detection of luk-PV (4), and antimicrobial susceptibility testing (Neo-Sensitabs), as well as microarray analysis (18, 19).Clinical and epidemiological information was obtained consecutively (17). Infections were categorized into four different groups: import, hospital associated, community associated, and health care associated with community onset (14).Where appropriate, statistical significance (P < 0.05) was assessed using the Mann-Whitney test or Fischer''s exact test.Between 1999 and 2006, 80 MRSA isolates from Denmark had USA300 PFGE profiles (50 representative PFGE profiles are shown in Fig. ​Fig.1).1). However, by spa typing, two different spa types, t008 (11-19-12-21-17-34-24-34-22-25 [n = 38]) and the single repeat variant t024 (11-12-21-17-34-24-34-22-25 [n = 42]), both belonging to ST8, were identified (the extra repeat [19] is shown in boldface). All isolates typed as SCCmec IVa. However, significant genetic, epidemiological, and clinical differences were found, as shown in Tables ​Tables11 and ​and2.2. Patients infected with t008 isolates were significantly younger than patients infected with t024 isolates (P < 0.01). Patients acquired t008 MRSA in the community (50%) or through travel abroad (21%), and infections were predominantly SSTIs (94%). In contrast, the majority of patients with t024 MRSA were either hospitalized (26%) or had health care-associated risk factors (38%), and they presented with SSTIs (64%) but, also, a larger variety of infections, including fatal respiratory tract infections (11%) and operation- and procedure-related infections (11%). Remarkably, no t024 MRSA cases were imported.Open in a separate windowFIG. 1.Unweighted pair group method with arithmetic mean (UPGMA) dendrogram of SmaI PFGE profiles from Danish MRSA isolates (2000 to 2005) constructed by the use of Dice determinations (optimization, 1.0%; tolerance, 2.1 to 3.1%). The reference strains are USA300 and USA500. Note that t008 and t024 isolates do not cluster separately by PFGE.

TABLE 1.

Bacteriological, epidemiological, and clinical data obtained for patients infected or colonized with either t008 or t024 in Denmark (1999 to 2005)

Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of SmaI Macrorestriction Fragments: a Multicenter Study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.The use of electric field pulsing techniques in conjunction with agarose gel electrophoresis for discrimination of large DNA molecules was introduced by Schwarz and Cantor in 1984 (9). During the past decade the methodology has been adapted and improved by various research groups to the point that pulsed-field gel electrophoresis (PFGE) for bacterial strain typing is now utilized with relative ease in a variety of laboratories (1). The combination of contour-clamped homogeneous field electrophoresis and PFGE for the molecular analysis of Staphylococcus aureus has been reported since the late 1980s (7, 19). At present, PFGE is considered to have both the reproducibility and resolving power of a standard technique for the epidemiological typing of bacterial isolates (10, 15).Molecular typing systems can identify different strains within a species, generating data useful for taxonomic or epidemiologic purposes (10, 14). A frequently observed shortcoming of typing systems in general is their lack of reproducibility: most typing systems do not provide a definitive strain identification, which is usually due to the variability of the technique and the lack of large databases containing fragment patterns from a wide variety of organisms to which unknowns can be compared. These problems were recently described in detail for two molecular typing systems. A multicenter study on random amplification of polymorphic DNA for discrimination of S. aureus strains revealed a lack of interlaboratory reproducibility among the banding patterns generated by the participating centers, although the epidemiological interpretation of the data was similar for all the centers involved (16). For PFGE, a similar lack of interlaboratory reproducibility of patterns was observed, although the interpretation of the experimental data also differed per participating center (2). The latter study analyzed 12 different methicillin-resistant S. aureus (MRSA) strains with different techniques optimized in each center and different sources and types of equipment. Since interlaboratory discrepancies with respect to classification of the strains were observed, the study concluded that there is a clear need for standardization of the technique, including the construction of a panel of reference strains to assist the individual researcher in the optimization of the PFGE protocol.The aim of the present study was to compare the fragment patterns of a well-defined collection of MRSA isolates in 12 laboratories using in-house and a standard set of PFGE parameters to determine whether standardization of experimental parameters (DNA preparation and switching protocols) would improve intercenter reproducibility of PFGE analysis.     

Impact of Strain Typing Methods on Assessment of Relationship between Paired Nares and Wound Isolates of Methicillin-Resistant Staphylococcus aureus

The anterior nares are the site of choice for the Veterans Administration methicillin-resistant Staphylococcus aureus (MRSA) surveillance program; however, a correlation between nares colonization and concomitant wound infections has not been well established. The purpose of this study was 3-fold: to determine the relatedness of MRSA isolates from 40 paired wound and nares specimens by four different strain typing methods, to determine concordance of typing methods, and to establish a baseline of MRSA types at this medical center. Isolates were typed by repetitive PCR (rep-PCR) (DiversiLab System; DL) and SpectraCell Raman analysis (SCRA) (commercially available methods that can be performed within a clinical lab), pulsed-field gel electrophoresis (PFGE), and an antibiotic susceptibility profile (AB). Whole-genome optical mapping (WGM) (OpGen, Inc.) was performed on selected isolates. All methods agreed that 26 pairs were indistinguishable and four pairs were different. Discrepant results were as follows: 4 where only SCRA was discordant, 3 where only AB was discordant, 2 where both DL and AB were discordant, and 1 where both DL and SCRA were discordant. All WGM agreed with PFGE. After discrepancy resolution, 80% of the pairs were indistinguishable and 20% were different. A total of 56% of nares results were nonpredictive if negative nares and positive wound cultures are included. Methods agreed 85 to 93% of the time; however, congruence of isolates to a clade was lower. Baseline analysis of types showed that 15 pairs were unique to single patients (30 strains, 38%; 47% of the matching pairs). Twenty-five strains (30%) represented a single clade identical by PFGE, SCRA, and DL, decreasing specificity. Typing method and institutional type frequency are important in assessing MRSA strain relatedness.