Comparison of Typing Results Obtained for Methicillin-Resistant Staphylococcus aureus Isolates with the DiversiLab System and Pulsed-Field Gel Electrophoresis

We compared the results of typing methicillin-resistant Staphylococcus aureus (MRSA) isolates using the DiversiLab system (DL) to the results obtained using pulsed-field gel electrophoresis (PFGE). One hundred five MRSA isolates of PFGE types USA100 to USA1100 and the Brazilian clone, from the Centers for Disease Control and Prevention (CDC) and Project ICARE strain collections, were typed using DL. In addition, four unique sets of MRSA isolates from purported MRSA outbreaks that had been previously typed by DL, each consisting of six isolates (where five isolates were classified as indistinguishable by DL and one was an unrelated DL type) were typed by PFGE. DL separated the 105 MRSA isolates of known USA types into 11 clusters and six unique banding patterns. DL grouped most of the USA100, USA200, and USA1100 isolates into unique clusters. Multilocus sequence type 8 isolates (i.e., USA300 and USA500) often clustered together at >95% similarity in DL dendrograms. Nevertheless, USA300 and USA500 DL patterns could be distinguished using the pattern overlay function of the DL software. Among the hospital outbreak clusters, PFGE and DL identified the same “unrelated” organism in three of four sets. However, PFGE showed more pattern diversity than did DL, suggesting that two of the sets were less likely to represent true outbreaks. In summary, DL is useful for screening MRSA isolates to rule out potential outbreaks of MRSA in hospitals, but PFGE provides better discrimination of potential outbreak strains and is more useful for confirming strain relatedness and specific USA types.Although pulsed-field gel electrophoresis (PFGE) is often considered the gold standard for typing methicillin-resistant Staphylococcus aureus (MRSA) isolates for epidemiologic studies (8, 12, 13), PFGE requires several days to complete and the results are often difficult for inexperienced users to interpret. On the other hand, DNA sequence-based methods, such as spa typing, which has also been shown to be useful for epidemiologic studies of MRSA (3), are not practical for many clinical laboratories in the United States, which lack access to DNA sequencing facilities. An alternative strain typing method, which is available commercially, is the DiversiLab typing system (DL) (bioMérieux, Inc., Durham, NC), which uses the presence of DNA repetitive elements present in the organism''s genome to determine the genetic relatedness of bacterial and fungal isolates (4-6, 9, 18). DL has been used successfully in several MRSA typing studies to distinguish sporadic from outbreak-related isolates and is noted to be more rapid to perform and easier to learn than PFGE (14, 15). Agreement between DL clusters of organisms and USA PFGE types, as defined by McDougal et al. (12), was reported for five well-defined U.S. outbreaks, although specific data were not shown (14). However, a recent study of representative MRSA strains from the Harmony collection in Europe concluded that while DL, PFGE, and multilocus sequence typing (MLST) provided concordant classification of strains, PFGE showed a higher level of strain discrimination than either DL or MLST (17). Thus, whether DL can differentiate accurately among USA types remains an open question.The goal of this study was to use DL to characterize a series of MRSA isolates of known PFGE types from U.S. hospitals to determine whether DL could (i) differentiate among PFGE types USA100 through USA1100, (ii) identify DL banding patterns that correlated with specific USA types, and (iii) differentiate contemporary outbreak-related MRSA isolates from sporadic isolates collected from U.S. hospitals.     

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.     

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.     

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.     

Differences between Two Clinical Staphylococcus capitis Subspecies as Revealed by Biofilm,Antibiotic Resistance,and Pulsed-Field Gel Electrophoresis Profiling

Coagulase-negative staphylococci have been identified as major causes of late-onset neonatal bacteremia in neonatal intensive care units. Sixty isolates of Staphylococcus capitis obtained from blood cultures of neonates between 2000 and 2005 were examined in this study. Biochemical analysis confirmed that 52 of these isolates belonged to the subsp. urealyticus, and the remaining 8 belonged to the subsp. capitis. Isolates of the predominant subsp. urealyticus clones were characterized by their resistance to penicillin, erythromycin, and oxacillin and their biofilm formation ability, whereas subsp. capitis isolates were generally antibiotic susceptible and biofilm negative. Pulsed-field gel electrophoresis (PFGE) after SacII digestion separated the 60 isolates into five major clusters. Sequence analysis showed that, in S. capitis, the ica operon plus the negative regulator icaR was 4,160 bp in length. PCRs demonstrated the presence of the ica operon in all isolates. Further analysis of five isolates (two biofilm-positive subsp. urealyticus, one biofilm-negative subsp. urealyticus, and two biofilm-negative subsp. capitis) revealed that the ica operons were identical in all of the biofilm-positive subsp. urealyticus strains; however, the biofilm-negative isolates showed variations. The distinctive phenotypic and genotypic characteristics revealed by this study may affect the epidemiology of the two subspecies of S. capitis in the clinical setting. These results may provide a better understanding of the contribution of these two species to bloodstream infections in neonates.     

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)

Epidemiological Study of a Food-Borne Outbreak of Enterotoxigenic Escherichia coli O25:NM by Pulsed-Field Gel Electrophoresis and Randomly Amplified Polymorphic DNA Analysis

This study investigated the applicability of molecular epidemiological techniques to the identification of the causal agent of an outbreak of diarrhea caused by ingestion of food contaminated with enterotoxigenic Escherichia coli (ETEC). The outbreak occurred at four elementary schools in July 1996 and affected more than 800 people. Illness was most strongly associated with eating tuna paste (relative risk, 1.79; 95% confidence interval = 1.16 to 2.79; P = 0.0001). To evaluate the epidemiological characteristics of the pathogen, the DNAs from numerous isolated ETEC strains were subjected to randomly amplified polymorphic DNA analysis, pulsed-field gel electrophoresis of nuclease S1-treated plasmid DNA, and analysis of genomic DNA restriction fragment length polymorphisms. All ETEC isolates were of the O25:NM (nonmotile) serotype, which carries a heat-stable enterotoxin Ib gene. Genotypic analysis demonstrated that the strains isolated from the patients at all four schools were identical. The isolates of ETEC O25:NM obtained from the tuna paste that had been served for lunch at these schools were genetically indistinguishable from those isolated from the patients. Results suggest that this outbreak was food borne. The molecular biology-based epidemiological techniques used in this study were useful in characterizing the causal agent in this food-borne epidemic.     

Outbreak of Staphylococcus schleiferi Wound Infections: Strain Characterization by Randomly Amplified Polymorphic DNA Analysis, PCR Ribotyping, Conventional Ribotyping, and Pulsed-Field Gel Electrophoresis

Within a 1-year period, six surgical-site infections (SSI) caused by Staphylococcus schleiferi were observed in the department of cardiac surgery of Ignatius Hospital, Breda, The Netherlands. Since outbreaks caused by this species of coagulase-negative staphylococci have not been described before, an extensive environmental survey and a case control study were performed in combination with molecular typing of the causative microorganism in order to identify potential sources of infection. Variability, as detected by four different genotyping methods (random amplification of polymorphic DNA [RAPD], conventional and PCR-mediated ribotyping, and pulsed-field gel electrophoresis [PFGE] of DNA macro restriction fragments), appeared to be limited both among the clinical isolates and among several control strains obtained from various unrelated sources. Among unrelated strains, RAPD and PCR-mediated ribotyping identified two types only, whereas seven different types were identified in a relatively concordant manner by conventional ribotyping and PFGE. The latter two procedures proved to be the most useful tools for tracking the epidemiology of S. schleiferi. Four of the outbreak-related strains were identical by both methods, and two isolates showed limited differences. In the search for a potential source of S. schleiferi infection, two slightly different PFGE types were encountered on several occasions in the nose of a single surgeon. These strains were, however, clearly different from the outbreak type. In contrast, S. schleiferi cultures remained negative for two persons identified on the basis of case control analysis. It was demonstrated that SSI caused by S. schleiferi had a clinical impact for patients comparable to that of a wound infection caused by Staphylococcus aureus. This report describes the first well-documented outbreak of S. schleiferi infection. A source of the outbreak was not detected.     

Genotypic Characterization of Salmonella enteritidis Phage Types by Plasmid Analysis, Ribotyping, and Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) was used to resolve XbaI and SpeI macrorestriction fragments from 60 defined phage type (PT) reference strains of Salmonella enteritidis. The level of discrimination was compared to that afforded by plasmid profile analysis and ribotyping. Twenty-eight distinct XbaI pulsed-field profiles (PFPs) were observed, although a single type, PFP X1, predominated. Absence of the 57-kb spv-associated fragment was observed for three PT reference strains, and the profile was designated PFP X1A. The XbaI macrorestriction profiles of a further four PT reference strains were altered by the presence of plasmid-associated bands. Twenty-six SpeI-generated PFPs (plus one subtype) were observed for the same strains. No SpeI fragment corresponding to the 38-MDa serovar-specific plasmid was detected. The distribution of XbaI and SpeI profiles did not always correspond, producing a total of 32 combined PFPs for the 60 PT reference strains. This compared with a total of 18 different plasmid profiles and three PvuII ribotypes generated by the same strains. The results of this study indicate that PFGE may offer an improved level of discrimination over other genotypic typing methods for the epidemiological typing of S. enteritidis.     

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.     

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.     

DNA heterogeneity of Staphylococcus aureus strains evaluated by SmaI and SgrAI pulsed-field gel electrophoresis in patients with impetigo

To our knowledge, no studies have previously been carried out on the heterogeneity and intrafamily colonization of impetigo Staphylococcus aureus strains obtained by powerful discriminating methods such as pulsed-field gel electrophoresis (PFGE). To explore this topic, macrorestriction patterns of S. aureus strains were analyzed after SmaI and SgrAI digestion. The two enzymes provided superimposable results. A total of ninety-seven S. aureus strains was found in the 26 families whose lesions and nasal and pharyngeal samples were examined. There were 39 strains which were different by PFGE, and of these, 24 were found in the lesions. Although 85% of impetigo patients showed nasal colonization and 58% showed pharyngeal colonization, only 54% of the patients had the same PFGE strain in the lesion and in the nose, and 35% in the lesion and the pharynx. In half of the 26 families, at least one member (mother, father, or relative) presented a S. aureus strain identical, by PFGE, to strains isolated in patients' lesions. Nineteen percent of mothers, 15% of fathers, and 19% of the other relatives presented nasal colonization with strains identical to those isolated in the children's lesions. Lesional strains showed higher antimicrobial resistance than nonlesional isolates.     

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.     

Genetic Features of Streptococcus agalactiae Strains Causing Severe Neonatal Infections, as Revealed by Pulsed-Field Gel Electrophoresis and hylB Gene Analysis

A collection of 114 independent Streptococcus agalactiae strains, including 54 strains isolated from the cerebrospinal fluid (CSF) samples of neonates and 60 strains from asymptomatic patients, was characterized by pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI and by PCR analysis of the hylB gene. All strains were previously studied by multilocus enzyme electrophoresis (MLEE) (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576-2581, 1995). Among these 114 strains, there were 92 PFGE patterns. Eleven genetic groups (A to K) were identified with 38% divergence. A more homogeneous group (PFGE group A) was defined, consisting of 73% of the strains previously identified as belonging to a particular MLEE phylogenetic group. A 162-kb fragment was identified as a marker of strains that invaded the central nervous system of neonates. It was detected in 69% of the PFGE patterns obtained with CSF isolates and in only 1.8% of the PFGE patterns obtained with carrier strains. The hylB gene encoding hyaluronate lyase was amplified for all strains in our collection. Ten of 15 isolates belonging to an MLEE subgroup, previously described as being likely to cause invasive infection, had an insertion in the hylB gene (IS1548).     

Proof of Principle for Successful Characterization of Methicillin-Resistant Coagulase-Negative Staphylococci Isolated from Skin by Use of Raman Spectroscopy and Pulsed-Field Gel Electrophoresis

Coagulase-negative staphylococci (CNS) are among the most frequently isolated bacterial species in clinical microbiology, and most CNS-related infections are hospital acquired. Distinguishing between these frequently multiple-antibiotic-resistant isolates is important for both treatment and transmission control. In this study we used isolates of methicillin-resistant coagulase-negative staphylococci (MR-CNS) that were selected from a large surveillance study of the direct spread of MR-CNS. This strain collection was used to evaluate (i) Raman spectroscopy as a typing tool for MR-CNS isolates and (ii) diversity between colonies with identical and different morphologies. Reproducibility was high, with 215 of 216 (99.5%) of the replicate samples for 72 isolates ending up in the same cluster. The concordance with pulsed-field gel electrophoresis (PFGE)-based clusters was 94.4%. We also confirm that the skin of patients can be colonized with multiple MR-CNS types at the same time. Morphological differences between colonies from a single patient sample correlated with differences in Raman and PFGE types. Some morphologically indistinguishable colonies revealed different Raman and PFGE types. This indicates that multiple MR-CNS colonies should be examined to obtain a complete insight into the prevalence of different types and to be able to perform an accurate transmission analysis. Here we show that Raman spectroscopy is a reproducible typing system for MR-CNS isolates. It is a tool for screening variability within a collection of isolates. Because of the high throughput, it enables the analysis of multiple colonies per patient, which will enhance the quality of clinical and epidemiological studies.Coagulase-negative staphylococci (CNS) are among the most frequently isolated bacterial species in clinical microbiology. They colonize the human skin, throat, nose, and/or gut and represent a major part of the normal bacterial flora of healthy people (15, 20). Although CNS have long been regarded as nonpathogenic, they have now been recognized as relevant opportunistic pathogens (23).Most CNS-related infections are hospital acquired. Since many nosocomial isolates are able to form biofilms on indwelling devices such as intravascular catheters, the eradication of CNS is difficult, and the removal of the catheter or device can be the only effective intervention. Bloodstream infections (BSI) caused by these organisms are frequently related to the use of medical devices. They account for significant morbidity and mortality, especially in immunocompromised individuals and neonates (16, 17, 20).A likely source of CNS isolates involved in catheter-related infections appears to be the skin flora. Several studies indicate that subtle morphological differences can be found among CNS colonies cultured from infected catheters, suggesting that multiple CNS types are present (3, 5, 18).Furthermore, it has been shown that CNS bacteria can be transmitted by hospitalized patients and health care workers (1, 13, 21). Isolates involved in transmission are often characterized by their resistance to many commonly used antibiotics, including methicillin (6, 9, 14). Although several studies suggest that colonized patients and health care workers should be considered reservoirs for these microorganisms, sometimes too little attention is paid to the control of these multiresistant isolates (7, 8, 23).Distinction between clinically significant and contaminating isolates as well as determining possible transmission routes in infection prevention and surveillance studies can be important. This, however, requires a more detailed characterization of isolates using an appropriate typing method. The most widely used typing method for outbreak analysis and surveillance of CNS is pulsed-field gel electrophoresis (PFGE). However, this technique is laborious and has a long turnaround time and low sample throughput, which makes it unsuitable for routine use and large-scale clinical studies. Currently, the “gold standard” to assess phylogenic relatedness between isolates is multilocus sequence typing (MLST). Recently, Raman spectroscopy has been reported to be a powerful tool for the identification of microorganisms at the species and subspecies levels. The technique is fast and easy to use. Raman spectra of bacteria are a representation of their overall molecular composition and can be used as strain-specific spectroscopic fingerprints (10, 12, 22). By using a collection of Staphylococcus aureus isolates, it was shown that the typing results obtained are comparable to those obtained by PFGE (22). Highly similar groupings compared to amplification fragment length polymorphism (AFLP) were previously documented for a collection of Acinetobacter isolates (10).In this study we used a collection of methicillin-resistant coagulase-negative staphylococci (MR-CNS) to illustrate that Raman spectroscopy can be used as a rapid and reproducible typing technique for MR-CNS isolates. We confirm that the skin of hospital patients can be colonized with multiple MR-CNS types, and therefore, screening for variability is essential for accurate transmission analysis.     

Improved Identification of Epidemiologically Related Strains of Salmonella enterica by use of a Fusion Algorithm Based on Pulsed-Field Gel Electrophoresis and Multiple-Locus Variable-Number Tandem-Repeat Analysis

Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) are used to assess genetic similarity between bacterial strains. There are cases, however, when neither of these methods quantifies genetic variation at a level of resolution that is well suited for studying the molecular epidemiology of bacterial pathogens. To improve estimates based on these methods, we propose a fusion algorithm that combines the information obtained from both PFGE and MLVA assays to assess epidemiological relationships. This involves generating distance matrices for PFGE data (Dice coefficients) and MLVA data (single-step stepwise-mutation model) and modifying the relative distances using the two different data types. We applied the algorithm to a set of Salmonella enterica serovar Typhimurium isolates collected from a wide range of sampling dates, locations, and host species. All three classification methods (PFGE only, MLVA only, and fusion) produced a similar pattern of clustering relative to groupings of common phage types, with the fusion results being slightly better. We then examined a group of serovar Newport isolates collected over a limited geographic and temporal scale and showed that the fusion of PFGE and MLVA data produced the best discrimination of isolates relative to a collection site (farm). Our analysis shows that the fusion of PFGE and MLVA data provides an improved ability to discriminate epidemiologically related isolates but provides only minor improvement in the discrimination of less related isolates.Salmonellosis is one of the most common food-borne diseases in the United States (5). Consequently, it is important to understand how Salmonella strains disseminate within and between reservoirs and environments. Many molecular typing tools have been used for this purpose (11). Of these methods, pulsed-field gel electrophoresis (PFGE) is considered by many to be the gold standard for strain typing, and variable-number tandem-repeat (VNTR) assays are powerful alternative or complementary typing tools (3, 22). Both methods offer a high degree of genetic resolution for strain typing, depending on several factors.PFGE involves separating chromosomal DNA macro-restriction fragments by size, and strains are discriminated based on the resulting band pattern observed after electrophoresis has been completed. It is one of the most reproducible and highly discriminatory typing techniques and has been widely and successfully used for a variety of Salmonella enterica serovars (12, 15); for many situations, PFGE is capable of discriminating between closely related strains. In addition, the use of the assay to analyze different serovars does not require a great deal of modification, as might be required with procedures that are dependent on PCR. Difficulties arise when strains are very closely related (i.e., poor discrimination [18, 27]) or when bands comigrate in the gel or identically sized bands represent completely different fragments of chromosomal DNA and thereby produce spurious matches (6). These complications are more pronounced when a large number of bands are generated by the restriction digest (4). In addition, while band patterns convey a crude degree of genetic relatedness, a large number of independent restriction digests would be needed to infer an accurate phylogenetic relationship (6).Multiple-locus variable-number tandem-repeat analysis (MLVA) is a PCR-based technique that relies on the amplification of chromosomal or plasmid DNA that encompasses short tandem repeats of a DNA sequence. The tandem repeats are prone to higher-than-background mutation rates due to DNA strand slippage during replication (23), and thus, the amplified fragments will vary in length depending on the number of repeats harbored at a given locus. Different fragment lengths are tallied either as the total length (base pairs) or the estimated number of repeat units, and each discretely sized fragment is considered a unique “allele” for the locus under investigation. Because of the relatively high mutation rate, strains can accumulate distinctive allele patterns within a relatively short period of time (5). Furthermore, the technique can be multiplexed and automated and is conducive to rapid and relatively high-throughput strain typing. MLVA assays are relatively robust (5, 15-17) and, while not perfect, they can provide phylogenetic information even with a limited number of loci (13, 18). While access to a sequenced genome dramatically speeds the ability to establish new assays (3), it is not a requisite to assay development. The primary limitations of the technique include the potential need for a new set of loci for every species or serovar under investigation and the fact that some loci are very “unstable” and can “disappear” from some strains or lineages; this produces the equivalent of an uninformative “null” allele. Mutation rates can also vary between loci (5, 24, 25); if ignored, this factor can introduce bias into comparative analyses.Clearly, PFGE and MLVA offer different technical and interpretive advantages and disadvantages, but it is important to emphasize that the nature of the methodological and interpretive errors is independent between the techniques. For example, errors due to comigration of bands for PFGE are independent of band size estimation errors for MLVA because differences in MLVA band size are not detectable using PFGE and macro-restriction fragments are generally independent of tandem-repeat sequences. Provided that most of the experimental variation from these two methods is uncorrelated (i.e., independent), it is possible to combine results from the two methods to produce improved estimates (8), and this premise underlies the current study.Our objective was to determine whether combining the information obtained from both PFGE and MLVA assays produces more rigorous and discriminatory analyses of bacterial pathogens, such as Salmonella. Two sets of Salmonella enterica isolates were used in this study; one set included serovar Typhimurium isolates from a wide range of sampling dates, locations, and host species while the other set included a group of serovar Newport isolates collected over a limited geographic and temporal scale for a single host species. The results of the different typing methods were assessed by comparison with those of phage-typing assays (serovar Typhimurium) and with known epidemiological relationships (serovar Newport). To interpret the MLVA data, we employed a metric that incorporates a stepwise-mutation model, and to interpret the PFGE data, we employed Dice coefficients to construct distance matrices. Our analysis shows that the fusion of the two typing methods provides an improved ability to discriminate between isolates when PFGE and MLVA separately provide partial but incomplete discrimination between strains with a high degree of probable genetic similarity.     

Molecular Analysis by Pulsed-Field Gel Electrophoresis and Antibiogram of Streptococcus pneumoniae Serotype 6B Isolates from Selected Areas within the United States

Fifty-eight clinical isolates of Streptococcus pneumoniae serotype 6B, including 16 from Alaska, 14 from Arizona, 11 from Washington, and 17 from seven additional states, were analyzed. The antibiograms of these isolates were assigned to 10 antibiotic profiles based on their susceptibilities to penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. Thirty-two (55%) of these isolates were penicillin nonsusceptible, while 21 (36%) were intermediate or resistant to three or more antibiotics. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by pulsed-field gel electrophoresis (PFGE). The ApaI and SmaI PFGE patterns were combined, and 13 of the 16 Alaskan isolates showed indistinguishable PFGE patterns. One other isolate exhibited highly related ApaI and SmaI PFGE patterns, differing by only one band after restriction with ApaI. Among the 14 isolates from Arizona, 1 was indistinguishable from the predominant ApaI and SmaI PFGE patterns seen in the Alaskan isolates; 5 others were highly related (±1 band after cutting with either enzyme) to the Alaskan isolates, suggesting a common ancestral origin. Of the remaining eight isolates, six additional ApaI plus SmaI PFGE patterns were observed. The 28 isolates from the various contiguous states had 22 ApaI plus SmaI PFGE patterns. No correlations were found between specific PFGE patterns, antibiograms, dates of isolation, or geography. The serotype 6B isolates across the contiguous United States were genetically diverse, while the 6B isolates from Alaska appeared to be much less diverse.     

Comparison of Pulsed-Gel Electrophoresis and a Commercial Repetitive-Element PCR Method for Assessment of Methicillin-Resistant Staphylococcus aureus Clustering in Different Health Care Facilities

Pulsed-field gel electrophoresis (PFGE) is a common method used to type methicillin-resistant Staphylococcus aureus (MRSA) in nosocomial investigations and epidemiological studies but is time-consuming and methodologically challenging. We compared typing results obtained using a commercial repetitive-element PCR (rep-PCR) system with PFGE in a sample of 86 unique MRSA isolates recovered from subjects in an academic referral hospital and two nursing homes in the same geographic region. Both methods reliably assigned isolates to the same Centers for Disease Control and Prevention (CDC) pulsotype. PFGE was significantly more discriminatory (Simpson''s index of diversity, 0.92 at the 95% strain similarity threshold) than the commercial rep-PCR system (Simpson''s index of diversity, 0.58). The global (adjusted Rand coefficient, 0.10) and directional congruence (adjusted Wallace coefficient_{repPCR→PFGE} = 0.06; adjusted Wallace coefficient_{PFGE→repPCR} = 0.52) between the two methods was low. MRSA strains recovered from study nursing homes that were clonal when typed by the commercial rep-PCR method were frequently noted to be genetically distinct when typed using PFGE. These data suggest that the commercial rep-PCR has less utility than PFGE in small-scale epidemiological assessments of MRSA in health care settings.