Niche-specific association of Aeromonas ribotypes from human and environmental origin

A total of 88 Aeromonas isolates from distinct locations and sources (39 from extraintestinal infections, 31 from diarrhoeic, ten from non-diarrhoeic faeces, all human, and eight from fresh water) were subjected to phenospecies identification, serotyping, ribotyping and detection of some virulence markers. The strains belonged to four different phenospecies marked by 19 O serogroups and 38 ribotypes. No strong correlation between these parameters was found, and no group, as defined by the typing methods, could be characterized with a particular set of virulence markers. There was a clear association of ribotypes with the source of the strains. Cluster analysis allowed the identification of a complex of ribotypes belonging to distinct but related sources, including clinical and environmental isolates. These results suggest that ribotyping may be an epidemiological tool suitable for the study of Aeromonas infections.

Molecular typing of IberoAmerican Cryptococcus neoformans isolates

A network was established to acquire basic knowledge of Cryptococcus neoformans in IberoAmerican countries. To this effect, 340 clinical, veterinary, and environmental isolates from Argentina, Brazil, Chile, Colombia, Mexico, Peru, Venezuela, Guatemala, and Spain were typed by using M13 polymerase chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphsm analysis with Hhal and Sau961 in a double digest. Both techniques grouped all isolates into eight previously established molecular types. The majority of the isolates, 68.2% (n=232), were VNI (var. grubii, serotype A), which accords with the fact that this variety causes most human cryptococcal infections worldwide. A smaller proportion, 5.6% (n=19), were VNII (var. grubii, serotype A); 4.1% (n=14), VNIII (AD hybrid), with 9 isolates having a polymorphism in the URA5 gene; 1.8% (n=6), VNIV (var. neoformans, serotype D); 3.5% (n=12), VGI; 6.2% (n=21), VGII; 9.1% (n=31), VGIII, and 1.5% (n=5) VGIV, with all four VG types containing var. gattii serotypes B and C isolates.

rDNA-RFLP identification of Candida species in immunocompromised and seriously diseased patients

PCR was used to amplify a targeted region of the ribosomal DNA of 76 Candida spp. isolates from immunocompromised and seriously diseased patients. Thirty-seven strains isolated from different anatomical sites of 11 patients infected with HIV (Vitória, ES, Brazil), 26 isolates from patients under treatment at Odilon Behrens Hospital and 13 isolates from skin and urine samples from São Marcos Clinical Analysis Laboratory (Belo Horizonte, Brazil) were scored. Fragments of rDNA were amplified using primer pairs ITS1-ITS4, for the amplification of ITS1 and ITS2 regions, including the gene for the 5.8s subunit. Amplification resulted in fragments ranging in size from 350 to 950 bp. Amplicons were digested with eight restriction enzymes. A pattern of species-specificity among the different medically important Candida species could be identified following restriction digestion of the PCR products. Candida albicans was the species most frequently observed, except for the group of newborns under treatment at the Odilon Behrens Hospital and for the isolates from the clinical analysis laboratory. C. parapsilosis was the species most frequently observed in these two groups.

Ocorrência de tuberculose em um hospital psiquiátrico do interior de Goiás

Objective: To investigate the prevalence of infection, disease and eventual institutional outbreak of tuberculosis in a psychiatric hospital using the PPD test, as well as testing for mycobacteria in material collected from the respiratory tree and using molecular tracking technique based on insertion sequence 6110 (IS6110). Methods: Between February and August of 2002, PPD tests were given to 74 inpatients and 31 staff members at a psychiatric hospital in the city of Rio Verde, located in the state of Goiás, Brazil. In addition, respiratory tree material collected from the inpatients was submitted to testing for Mycobacterium tuberculosis. Results: Among the patients analyzed, mycobacteria were isolated from five (6.8%): four identified as M. tuberculosis and one as M. chelonae. The M. tuberculosis isolates were sensitive to isoniazid and rifampicin, and, when submitted to the restriction fragment length polymorphism/IS6110 technique, presented unique genetic profiles, totally distinct from one another, suggesting that all of the tuberculosis cases were due to endogenous reactivation. It was not possible to characterize this group of cases as an institutional outbreak. Performing the two-step tuberculin test in the patients, the infection rates were 23% and 31%, compared with 42% among staff members, who were submitted to the one-step test. Conclusion: The results indicate a high incidence of tuberculosis infection among inpatients and hospital staff, as well as a high occurrence of the disease among inpatients.

Transposon display supports transpositional activity of P elements in species of the saltans group of Drosophila

Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances between strains regarding the insertion polymorphism profile were estimated. Amplification of the P element based on copy number estimates was highly variable among the strains (D. sturtevanti, canonical 20.11, O-type 9.00; D. saltans, canonical 16.4, O-type 12.60 insertions, on average). The larger values obtained by TD compared to our previous data by Southern blotting support the higher sensitivity of TD over Southern analysis for estimating transposable element copy numbers. The higher numbers of the canonical P element and the greater divergence in its distribution within the genome of D. sturtevanti (24.8%) compared to the O-type (16.7%), as well as the greater divergence in the distribution of the canonical P element, between the D. sturtevanti (24.8%) and the D. saltans (18.3%) strains, suggest that the canonical element occupies more sites within the D. sturtevanti genome, most probably due to recent transposition activity. These data corroborate the hypothesis that the O-type is the oldest subfamily of P elements in the saltans group and suggest that the canonical P element is or has been transpositionally active until more recently in D. sturtevanti. © Indian Academy of Sciences.

Valor preditivo do potencial de implantação embrionário pela análise do perfil químico de meios de cultivo por espectrometria de massas (ESI-Q-ToF)

Objective: This case-control study analyzed mass spectrometry fingerprinting patterns of culture media samples used for embryo culture to predict embryo implantation. Methods: The culture medium harvested after embryo transfer of 22 embryos from 13 patients was used for the experiments. After embryo transfer, the remaining culture media were collected and samples were split in positive (n=8) and negative (n=14) implantation groups according to implantation outcomes (100% or 0% of implantation). Samples were individually diluted and injected directly to the Electrospray ionization (ESI) MS coupled to a Quadrupole Time-of-flight MS (Q-ToF-MS).Ions relative intensities of each spectrum were considered. Data analysis was conducted in MatLab 7.0 version using Partial Least Squares - Discriminant Analysis toolbox. Results: There were 3027 observed ions at 100% and 0% implantation groups by ESI-Q-ToF-MS. The statistical model could categorize the samples in two clusters, based on their positive and negative implantation outcomes. Less intense ions present in the mass spectra with statistical significance have contributed to the major differences to group distinction. Conclusions: Positive and negative implantation embryos showed a specific biochemical pattern present in culture media, which could be detected as a fast, simple and non-invasive way. This biochemical profile could help the selection of the most viable embryo, improving single embryo transfer and thus eliminating the risk and undesirable outcomes of multiple pregnancies. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

Evaluation of Xylella fastidiosa genetic diversity by fAFPL markers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Entwicklung und Anwendung molekularbiologischer Methoden zur Art- und Stamm-Identifizierung pro- und eukaryotischer Organismen

Das Wachstum von Milchsäurebakterien-Arten der Gattungen Lactobacillus, Pediococcus und Leuconostoc während der Weinfermentation kann durch die Bildung verschiedener Stoffwechselprodukte zu Weinfehlern führen. Um rechtzeitig Gegenmaßnahmen ergreifen zu können und einem Weinverderb vorzubeugen, bedarf es geeigneter Identifizierungsmethoden. Klassische mikrobiologische Methoden reichen oft nicht aus, um Mikroorganismen auf Art- und Stammniveau gezielt zu identifizieren. Wegen ihrer schnellen Durchführbarkeit und Zuverlässigkeit sind molekularbiologische Identifizierungsmethoden zur Kontrolle der mikrobiellen Flora während der Lebensmittelfermentierung in der heutigen Zeit unabdingbar. In der vorliegenden Forschungsarbeit wurden die 23S rRNA-Gensequenzen von neun Pediococcus-Typstämmen sequenziert, analysiert und phylogenetische Analysen durchgeführt. Zur Art-Identifizierung der Pediokokken wurden PCR-Primer generiert und ein Multiplex PCR System entwickelt, mit dem alle typischen Arten simultan in einer Reaktion nachgewiesen werden konnten. Die Ergebnisse der Multiplex PCR-Identifizierung von 62 Pediococcus-Stämmen aus Kulturensammlungen und 47 neu isolierten Stämmen aus Wein zeigten, dass einige Stämme unter falschen Artnamen hinterlegt waren, und dass P. parvulus im Weinanbaugebiet Rheinhessen weit verbreitet war. Die Fähigkeit der Pediococcus-Stämme zur Exopolysaccharid-Synthese wurde durch den Nachweis zweier Gene überprüft. Auf Basis der 23S rDNA-Sequenzen wurden rRNA-Sekundärstrukturen mit der neu entwickelten Software Structure Star generiert, die zum Auffinden von Zielbereichen für fluoreszenzmarkierte DNA-Sonden geeignet waren. Die Sequenzunterschiede zwischen den Pediococcus-Arten reichten aus, um zwei Gruppen durch Fluoreszenz in situ Hybridisierung differenzieren zu können. Die Verwendung unmarkierter Helfer-sonden verbesserte die Zugänglichkeit der Sonden an die rRNA, wodurch das Fluoreszenz-Signal verstärkt wurde. Um Milchsäurebakterien durch Denaturierende Gradienten Gel Elektrophorese differenzieren zu können, wurden Primer entwickelt, mit denen ein hochvariabler 23S rDNA-Bereich amplifiziert werden konnte. Die Nested Specifically Amplified Polymorphic DNA (nSAPD)-PCR wurde in der vorliegenden Arbeit zur Art- und Stamm-Differenzierung pro- und eukaryotischer Organismen angewandt. Es wurden vor allem weinrelevante Milchsäurebakterien der Gattungen Oenococcus, Lactobacillus, Pediococcus und Leuconostoc und Hefen der Gattungen Dekkera / Brettanomyces und Saccharomyces untersucht. Die Cluster-Analyse der Pediococcus-Typstämme führte zu einer unterschiedlichen Baum-Topologie im Vergleich zum phylogenetischen 23S rDNA-Stammbaum. Die Verwandtschaftsverhältnisse der untersuchten O. oeni-Stämme aus Starterkulturen konnten in Bezug auf eine frühere Cluster-Analyse reproduziert werden. Die Untersuchung von 40 B. bruxellensis-Stämmen aus rheinhessischen Weinproben zeigte eine Gruppierung der Stämme gemäß dem Ort der Probennahme. Beim Vergleich der Verwandtschaftsverhältnisse von Stämmen der Arten P. parvulus und B. bruxellensis, die aus denselben Weinproben isoliert wurden, konnte eine hohe Übereinstimmung der beiden Baum-Topologien beobachtet werden. Anhand der SAPD-PCR Untersuchung von Sekthefen aus Starterkulturen konnten alle Stämme der Art S. cerevisiae zugeordnet werden. Die nSAPD-PCR war darüber hinaus geeignet, um höhere Eukaryoten wie Weinreben zu differenzieren und es konnten die Verwandtschaftsverhältnisse von Mäusen und menschlichen Individuen durch Cluster-Analysen nachvollzogen werden. Mit Hilfe der Sequence Characterized Amplified Region (SCAR)-Technik wurden (n)SAPD-Marker in SCAR-Marker konvertiert. Die neu generierten SCAR-Primer konnten zur simultanen Art-Identifizierung von sieben weinschädlichen Milchsäurebakterien in einer Multiplex PCR erfolgreich eingesetzt werden. Die in dieser Arbeit entwickelten molekularbiologischen Identifizierungsmethoden können zum Beispiel in der mikrobiologischen Qualitätskontrolle Anwendung finden.

Identifizierung, Quantifizierung und Hemmung von ausgewählten Hefen im Wein

Hefen stellen einen großen und wichtigen Teil der Mikrobiota während der Weinbereitung dar, da ohne ihre alkoholische Fermentation die Umwandlung von Most und Wein nicht möglich wäre. Ferner ist es ihre Vielzahl an Stoffwechselprodukten, die dem Aroma des fertigen Weines eine zusätzliche Komplexität verleihen. Auf der anderen Seite steht durch den Metabolismus verschiedenster so genannter Wildhefen die Gefahr von Qualitätsabstufungen der Weine, was allgemein als „Weinfehler“ betrachtet wird. Ziel dieser Arbeit war zum einen die taxonomische Einordnung von Saccharomyces-Spezies, sowie die Quantifizierung und Hemmung von ausgewählten Wildhefen während der Weinbereitung.rnEin Teil dieser Arbeit umfasste die Identifizierung der nahverwandten Mitglieder der Saccharomyces sensu stricto-Gruppe. Durch den Einsatz des DNA-Fingerpinting-Systems SAPD-PCR konnten alle die Gruppe umfassenden Spezies anhand spezifischer Bandenmuster nachgewiesen werden, wodurch eine Einordnung dieser schwer zu differenzierenden Arten möglich war. Die Differenzierung zwischen den einzelnen Spezies war in jedem Fall deutlicher als dies die Sequenzierung der 5.8S rDNA und ihre flankierenden ITS-Regionen vermochte. Die SAPD-PCR zeichnete sich zudem durch eine geringe Muster-Varianz bei verschiedenen Stämmen einer Art aus und konnte zuverlässig unbekannte Stämme bestimmen und bereits hinterlegte Stämme neu klassifizieren. Zudem konnte mit Hilfe dieses Systems Hybride aus Saccharomyces cerevisiae und S. bayanus bzw. S. cerevisiae und S. kudriavzevii detektiert werden, wenn diese Hybride aus relativ gleichen genomischen Anteilen der Eltern bestanden. rnZusätzlich wurde ein quantitatives PCR-System entwickelt, um die Gattungen Saccharomyces, Hanseniaspora und Brettanomyces in Most und Wein detektieren und quantifizieren zu können. Die hierfür entwickelten Primer zeigten sich spezifisch für die untersuchten Arten. Durch die serielle Verdünnung definierter DNA-Mengen konnte für alle drei Systeme eine Kalibrierungskurve erstellt werden, mit Hilfe derer die tatsächlichen Quantifizierungen durchgeführt wurden. Die qPCR-Analyse lieferte ähnliche Zellzahlen wie Lebendzellzahl-Bestimmungen und wurde nicht von anderen Spezies und von Traubensaft gestört. Die maximal detektierbare Zellzahl betrug 2 x 107 Zellen/ml, während die minimale Detektionsgrenze je nach Art zwischen 1 x 102 Zellen/ml und 1 x 103 Zellen/ml lag. Allerdings konnte eine effektive DNA-Isolierung dieser geringen Zellzahlen nur erreicht werden, wenn die Zellzahl durch artfremde Hefen künstlich erhöht wurde. Die Analyse einer Most-Vergärung mit den drei Spezies zeigte schlussendlich, dass die quantitative PCR sicher und schnell Veränderungen und Sukzessionen detektiert und so ein geeignetes Mittel darstellt, um Populationsdynamiken während der Weinherstellung zu beobachten. rnDer letzte Teil dieser Arbeit befasste sich mit der Inhibierung von Schadhefen durch zellwand-hydrolysierende Enzyme. Es konnte hierbei eine endoglykosidisch wirkende β-1,3-Glucanase aus dem Bakterium Delftia tsuruhatensis isoliert werden. Diese besaß eine ungefähre Masse von 28 kDa, einen isolektrischen Punkt von ca. 4,3 und wirkte mit einer spezifischen Aktivität von 10 U/mg Protein gegen das Glucan Laminarin. Zudem zeigte das Enzym ein Temperaturoptimum von 50 °C und ein pH-Optimum bei pH 4,0. Weinparameter wie erhöhte Konzentrationen an Ethanol, Phenolen und Sulfit beeinflussten die Wirkung des Enzyms nicht oder nur wenig. Neben der allgemeinen Wirkung gegen β-1,3-Glucane konnte hier auch gezeigt werden, dass ebenso gut die β-1,3-Glucane in der Zellwand verschiedener Hefen hydrolysiert wurden. Fluoreszenz- und rasterelektronen-mikroskopische Aufnahmen von Hefezellen nach Inkubation mit der β-1,3-Glucanase zeigten zusätzlich die Zerstörung der Zelloberfläche der Hefen. Die lytische Wirkung des Enzyms wurde an verschiedenen weintypischen Hefen getestet. Hierbei zeigten sich stammspezifische Unterschiede in der Sensitivität gegenüber dem Enzym. Außerdem konnte festgestellt werden, dass sowohl Wachstumsphase als auch Medium der Hefen Einfluss auf deren Zellwand hat und somit auch auf die Wirkung des Enzyms.rn

Extensive clonal spread and extreme longevity in saw palmetto, a foundation clonal plant

The lack of effective tools have hampered our ability to assess the size, growth and ages of clonal plants. With Serenoa repens (saw palmetto) as a model, we introduce a novel analytical framework that integrates DNA fingerprinting and mathematical modelling to simulate growth and estimate ages of clonal plants. We also demonstrate the application of such life-history information of clonal plants to provide insight into management plans. Serenoa is an ecologically important foundation species in many Southeastern United States ecosystems; yet, many land managers consider Serenoa a troublesome invasive plant. Accordingly, management plans have been developed to reduce or eliminate Serenoa with little understanding of its life history. Using Amplified Fragment Length Polymorphisms, we genotyped 263 Serenoa and 134 Sabal etonia (a sympatric non-clonal palmetto) samples collected from a 20 X 20 m study plot in Florida scrub. Sabal samples were used to assign small field-unidentifiable palmettos to Serenoa or Sabal and also as a negative control for clone detection. We then mathematically modelled clonal networks to estimate genet ages. Our results suggest that Serenoa predominantly propagate via vegetative sprouts and 10000-year-old genets may be common, while showing no evidence of clone formation by Sabal. The results of this and our previous studies suggest that: (i) Serenoa has been part of scrub associations for thousands of years, (ii) Serenoa invasion are unlikely and (ii) once Serenoa is eliminated from local communities, its restoration will be difficult. Reevaluation of the current management tools and plans is an urgent task.

Extensive clonal spread and extreme longevity in saw palmetto, a foundation clonal plant

The lack of effective tools has hampered our ability to assess the size, growth and ages of clonal plants. With Serenoa repens (saw palmetto) as a model, we introduce a novel analytical frame work that integrates DNA fingerprinting and mathematical modelling to simulate growth and estimate ages of clonal plants. We also demonstrate the application of such life-history information of clonal plants to provide insight into management plans. Serenoa is an ecologically important foundation species in many Southeastern United States ecosystems; yet, many land managers consider Serenoa a troublesome invasive plant. Accordingly, management plans have been developed to reduce or eliminate Serenoa with little understanding of its life history. Using Amplified Fragment Length Polymorphisms, we genotyped 263 Serenoa and 134 Sabal etonia (a sympatric non-clonal palmetto) samples collected from a 20 x 20 m study plot in Florida scrub. Sabal samples were used to assign small field-unidentifiable palmettos to Serenoa or Sabal and also as a negative control for clone detection. We then mathematically modelled clonal networks to estimate genet ages. Our results suggest that Serenoa predominantly propagate via vegetative sprouts and 10000-year-old genets maybe common, while showing no evidence of clone formation by Sabal. The results of this and our previous studies suggest that: (i) Serenoa has been part of scrub associations for thousands of years, (ii) Serenoa invasions are unlikely and (ii) once Serenoa is eliminated from local communities, its restoration will be difficult. Reevaluation of the current management tools and plans is an urgent task.

Analysis of non-porcine isolates of Actinobacillus suis

Twenty-four Actinobacillus suis isolates obtained from several species of non-porcine mammals were compared to the representative porcine strains, ATCC 15557 (serotype O1) and H89-1173 (serotype O2), by O serotyping, DNA fingerprinting, PCR amplification of apxICA, apxIICA and apxIIICA toxin genes and by rrs (16S rRNA) gene sequencing. Only two strains, both equine, reacted with O1 antiserum while two others, one canine and the other feline, reacted with O2 antiserum. One equine strain reacted weakly with both antisera. No amplification of apx genes was found with the non-porcine O1 or the "not O1/O2" strains but amplification of the apxICA and apxIICA genes was observed with the two O2 strains. In addition, these two O2 strains had both BamHI and BglII fingerprints that were very similar to the porcine O2 reference strain, H89-1173 and rrs gene sequences that were identical to the A. suis reference strain ATCC 15557. Taken together, these data suggest that although many non-porcine A. suis isolates are not A. suis (sensu stricto), some isolates are genotypically as well as phenotypically similar to A. suis.

Molecular epidemiology of vancomycin-resistant Enterococcus faecium: a prospective, multicenter study in South American hospitals.

Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal cluster 17 (CC17). Enterococcal isolates were collected prospectively (2006 to 2008) from 32 hospitals in Colombia, Ecuador, Perú, and Venezuela and subjected to antimicrobial susceptibility testing. Genotyping was performed with all vancomycin-resistant E. faecium (VREfm) isolates by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. All VREfm isolates were evaluated for the presence of 16 putative virulence genes (14 fms genes, the esp gene of E. faecium [espEfm], and the hyl gene of E. faecium [hylEfm]) and plasmids carrying the fms20-fms21 (pilA), hylEfm, and vanA genes. Of 723 enterococcal isolates recovered, E. faecalis was the most common (78%). Vancomycin resistance was detected in 6% of the isolates (74% of which were E. faecium). Eleven distinct PFGE types were found among the VREfm isolates, with most belonging to sequence types 412 and 18. The ebpAEfm-ebpBEfm-ebpCEfm (pilB) and fms11-fms19-fms16 clusters were detected in all VREfm isolates from the region, whereas espEfm and hylEfm were detected in 69% and 23% of the isolates, respectively. The fms20-fms21 (pilA) cluster, which encodes a putative pilus-like protein, was found on plasmids from almost all VREfm isolates and was sometimes found to coexist with hylEfm and the vanA gene cluster. The population genetics of VREfm in South America appear to resemble those of such strains in the United States in the early years of the CC17 epidemic. The overwhelming presence of plasmids encoding putative virulence factors and vanA genes suggests that E. faecium from the CC17 genogroup may disseminate in the region in the coming years.

A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen.

Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm(+)) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (P

A trilocus sequence typing scheme for hospital epidemiology and subspecies differentiation of an important nosocomial pathogen, Enterococcus faecalis.

In this study, we present a trilocus sequence typing (TLST) scheme based on intragenic regions of two antigenic genes, ace and salA (encoding a collagen/laminin adhesin and a cell wall-associated antigen, respectively), and a gene associated with antibiotic resistance, lsa (encoding a putative ABC transporter), for subspecies differentiation of Enterococcus faecalis. Each of the alleles was analyzed using 50 E. faecalis isolates representing 42 diverse multilocus sequence types (ST(M); based on seven housekeeping genes) and four groups of clonally linked (by pulsed-field gel electrophoresis [PFGE]) isolates. The allelic profiles and/or concatenated sequences of the three genes agreed with multilocus sequence typing (MLST) results for typing of 49 of the 50 isolates; in addition to the one exception, two isolates were found to have identical TLST types but were single-locus variants (differing by a single nucleotide) by MLST and were therefore also classified as clonally related by MLST. TLST was also comparable to PFGE for establishing short-term epidemiological relationships, typing all isolates classified as clonally related by PFGE with the same type. TLST was then applied to representative isolates (of each PFGE subtype and isolation year) of a collection of 48 hospital isolates and demonstrated the same relationships between isolates of an outbreak strain as those found by MLST and PFGE. In conclusion, the TLST scheme described here was shown to be successful for investigating short-term epidemiology in a hospital setting and may provide an alternative to MLST for discriminating isolates.