607 research outputs found
High prevalence of clonally diverse spa type t026 staphylococcus aureus contaminating rural eggshells
Purpose. The presence of Staphylococcus aureus in poultry and poultry products, including eggs, increases its potential to enter the food chain, resulting in foodborne diseases. In this context, eggshell colonization by staphylococci may represent a risk factor. This study aimed to investigate the contamination of rural eggshell by S. aureus and to characterize the key features of the isolated strains. Methodology. Antibiotic resistance was assessed by disc diffusion. Resistant isolates were analysed by PCR for the identification of associated genetic determinants of resistance. PCR was also used to screen for the presence of genes coding for toxins, namely, sea, sec, sei, sem, seo and tst. The genetic characterization was extended by means of agr locus typing and spa typing. Results. 34 S. aureus were isolated. Macrolide-and tetracycline-resistant strains were prevalent. All strains were susceptible to oxacillin, cefoxitin and trimethoprim-sulfamethoxazole. PCR screening for genes encoding enterotoxins detected several virulence patterns, which, together with spa-typing and agr-locus typing, allowed cluster analysis and the description of novel clones. Conclusion. Continuous monitoring of staphylococci is needed also in rural or natural settings. Increasing the number of samples and expanding the geographical region will be needed to further extend the significance of the study
Genome-wide association study reveals a locus for nasal carriage of <i>Staphylococcus aureus</i> in Danish crossbred pigs
BACKGROUND: Staphylococcus aureus is an important human opportunistic pathogen residing on skin and mucosae of healthy people. Pigs have been identified as a source of human colonization and infection with methicillin-resistant Staphylococcus aureus (MRSA) and novel measures are needed to control zoonotic transmission. A recent longitudinal study indicated that a minority of pigs characterized by high nasal load and stable carriage may be responsible for the maintenance of S. aureus within farms. The primary objective of the present study was to detect genetic loci associated with nasal carriage of S. aureus in Danish crossbred pigs (Danish Landrace/Yorkshire/Duroc). RESULTS: Fifty-six persistent carriers and 65 non-carriers selected from 15 farms surveyed in the previous longitudinal study were genotyped using Illumina’s Porcine SNP60 beadchip. In addition, spa typing was performed on 126 S. aureus isolates from 37 pigs to investigate possible relationships between host and S. aureus genotypes. A single SNP (MARC0099960) on chromosome 12 was found to be associated with nasal carriage of S. aureus at a genome-wide level after permutation testing (p = 0.0497) whereas the association of a neighboring SNP was found to be borderline (p = 0.114). Typing of S. aureus isolates led to detection of 11 spa types belonging to the three main S. aureus clonal complexes (CC) previously described in pigs (CC9, CC30 and CC398). Individual carriers often harbored multiple S. aureus genotypes and the host-pathogen interaction seems to be independent of S. aureus genotype. CONCLUSION: Our results suggest it may be possible to select pigs genetically resistant to S. aureus nasal colonization as a tool to control transmission of livestock-associated MRSA to humans. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12917-015-0599-y) contains supplementary material, which is available to authorized users
A prospective methicillin resistant staphylococcus aureus typing system for infection control : design and effectiveness
The uptake of Gadomer-17, as probed by fast dynamic T(1) measurements, was used to assess the vascular permeability surface-area product per leakage volume of tissue (k(Tofts)) of human glioma xenografts implanted in mice. With this approach we could discriminate between two types of glioma xenograft lines with a known difference in the perfused vascular architecture and degree of hypoxia. The T(1) data were analyzed according to the Tofts-Kermode compartment model. The fast-growing E102 tumor demonstrated a homogeneous distribution of the vascular permeability surface area across the tumor (mean k(Tofts) value = 0.18 +/- 0.05 min(-1)). The slowly growing E106 tumor showed a more heterogeneous pattern. Three perfused tumor areas with differences in vascular permeability surface area could be distinguished: a well-perfused periphery with high k(Tofts) values (0.24 +/- 0.04 min(-1)), perfused capillaries inside the tumor with low k(Tofts) values (0.108 +/- 0.026 min(-1)), and perfused capillaries adjacent to necrotic regions with high k(Tofts) values (0.29 +/- 0.10 min(-1)). On a different series of tumors, the hypoxic fractions were measured, and these were significantly higher in E106 tumors (0.14 +/- 0.05) compared to tumors of the E102 line (0.03 +/- 0.02)
Genotypic and mass spectrometric comparative analysis of African and German Staphylococcus aureus isolates
Staphylococcus aureus (SA) is a worldwide distributed, opportunistic pathogen
colonizing 25-30% of the human population mostly without signs of infections.
However, if infections (e.g. wound-, organ infections, sepsis) occurs, those are often
associated with a significant morbidity and mortality. SA possesses a broad range of
pathogenicity-, virulence- and resistance factors, whose presence is partially
associated with specific phylogenetic developmental lines. Thus, the evaluation of
transmission of infections is essentially based on phylogenetic analytics. This requires
the development of reproducible, highly discriminatory typing methods for distinction
of different SA strains. Thus, reproducible, polymerase chain reaction and sequencing
based methods such as spa (SA protein A)-typing, multilocus sequencing typing
(MLST) up to whole genome sequencing were developed. In contrast to spa-typing
which is based on the sequencing of the single gene spa, MLST is based on the
sequencing of seven housekeeping genes defining different sequence types (STs)
which can be clustered to clonal complexes (CC). STs and CCs are today the grid for
the international SA nomenclature in epidemiologic studies. DNA microarrays (MA)
represents a special technique in which ST and CC are deduced from characteristic
hybridization patterns of numerous genloci which are compared to a database of a
large strain collection. In addition to the phylogenetic analysis the DNA MA hold a large
number of locis for adhesion, virulence, toxin and resistance genes and thus provide a
large amount of data. Comparison of multiple isolates investigated by DMA MA thus,
requires supplementary bioinformatic methods for interpretation and presentation.
Overall, there are plenty of moleculargenetic based studies of infection epidemiology
of SA. But, most of them referred to countries with a high level of microbiological
diagnostic. For Africa there is only very limited epidemiological data on SA (inclusive
methicillin-resistant SA [MRSA]) especially of non-nosocomial (this means not
acquired in hospital) SA (e.g. community-associated (CA) methicillin-resistant or
methicillin-susceptible SA [MRSA, MSSA]). In order to fill this knowledge gap, four
studies were performed as basis for this doctoral thesis investigating a) different
bioinformatic tools for analysis of the molecular epidemiology of SA, b) the
epidemiology of African and German MSSA/MRSA originating outside healthcare institutions and c) the applicability of mass spectrometry for analysis of SA
epidemiology.
For characterization of the bioinformatic tools the subtyping potential of spa-typing
versus DNA MA analysis were compared in a study with 46 nasal CA-MSSA and 46
CA-MRSA of infections, collected in the federal state of Saarland. Further, the results
were evaluated by three bioinformatic methods (splits graph, hierarchical
agglomerative clustering [HAC] and principal component analysis [PCA]). Finally, in
order to test the applicability of the DNA MA analytics in the African cohort, a relatively
small cohort of isolates of 52 Nigerian MSSA was analyzed by DNA MA and splits
graph method.
In a big, multicentric, African-German, cross-sectional geographic comparative
analysis, 1200 CA-SA isolates of healthy volunteers and patients of Sub-Saharan
Africa and Germany were subsequently analyzed by DNA MA. In addition, association
and comparative analyses of STs, CCs and gene composition of these isolates with
respect to geographic (Germany or Africa) or clinical/nasal origin (nasal/asymptomatic
or clinical/invasive) were performed. Finally, the potential applicability of identification
of CCs or spa–types of CA-SA of CC5, CC8, CC15, CC30, CC45, CC121 and
spa-types t002, t003, t504 based on their MALDI-TOF mass spectrometric profile, were
also analyzed.
Expected results on the epidemiology of CA-MRSA/MSSA were firstly gained by the
DNA MA study of the federal state of Saarland, showing a higher subtyping potential
of the DNA MA compared to spa-typing. Evaluation of the DNA MA data by the three
previously mentioned bioinformatic methods yielded a diverse assignment of CC5
isolates to 3 to 7 clusters with exception of 12 CC5 isolates that always clustered in
two identical clusters. It does not make these methods a failure but the use of different
mathematical algorithms analyzing hybridization pattern differences or similarities may
lead to minor differences. Further, MSSA displayed a high degree of diversity in
comparison to the CA-MRSA group, which was dominated by the CC5 Rhine-Hesse
epidemic strain (89%).
The further analysis of African MSSA of the study with Nigerian MSSA identified a
heterogeneous and divergent nature of the examined Nigerian MSSA and confirmed
that the DNA MA is a suitable genotyping method of African SA. The results of the
multicentric consortium study showed a total of 40 different CCs and STs, including 5
new STs. The majority, 15 of the 22 (68%) most common CC’s, were either predominant in Sub-Saharan Africa (Gabon, Mozambique, Tanzania) (CC80, CC88,
CC121, CC152) or Germany (CC22, CC30, CC45, CC398). The rate of MRSA carriers
in the African (3%) and German populations (1%) were very low, probably because the
investigation was limited to CA-SA. Analysis of gene profiles identified a high
prevalence of the Panton-Valentine leukocidin encoding genes lukF/S-PV in African
isolates. Generally, continent-specific differences for the CCs and the CA-SA gene
profiles could be identified. Furthermore, it became apparent that in addition to the
good first-line characterization by DNA MA, standardized, complex analytical methods
for association and epidemiological analyses are required.
The last investigation purpose, the evaluation of comparative mass spectra analysis
showed that only SA of CC121 were correctly identified based on their mass
spectrometric profile on the CC level, while no clear discrimination was achieved for
other CCs or spa-types.
Taken together, this work identified country specific differences in the distribution of
CCs, and specific genes of the examined regions, especially concerning only clinical
isolates. The work provide first clues to the genetic based reasons which may explain
the purported difference in clinical presentation and course of diseases caused by SA.
Additionally, it was shown that DNA MA in contrast to mass spectrometry is a suitable
tool for SA characterization e.g. in outbreak situations. However, it should be noted
that as specific requirement of a challenging DNA MA based phylogenetic analyses,
application of complex bioinformatic analysis methods are required for interpretation of
multiple isolates, and that the characteristics and algorithms of the chosen
bioinformatic method may result in slight differences in the data interpretation.Staphylococcus aureus (SA) ist ein weltweit verbreitetes, opportunistisches Pathogen,
dass 25-30% der Bevölkerung häufig symptomfrei kolonisiert. Treten jedoch invasive
Infektionen auf (z.B. Wund-, Organinfektion, Sepsis), so sind diese mit erheblicher
Morbidität und Mortalität assoziiert. SA besitzt ein breites Spektrum an Pathogenitäts-,
Virulenz- und Resistenzfaktoren, deren Vorhandensein teilweise mit besonderen
phylogenetischen Entwicklungslinien assoziiert ist. So basiert die Bewertung von
Infektionsübertragung und Ausbruchsgeschehen vielfach auch auf phylogenetischer
Analytik. Dies erfordert die Entwicklung reproduzierbarer Typisierungsverfahren, die die unterschiedlichen SA-Stämme auch ausreichend trennscharf charakterisieren
können. Daher wurden reproduzierbare, einheitlich auswertbare
Polymerasekettenreaktionen und Sequenzierungsmethoden wie spa (SA Protein A)-
Typisierung, Multilokussequenztypisierung (MLST), bis hin zur
Gesamtgenomsequenzierung entwickelt. Im Gegensatz zur spa-Typisierung,
basierend auf der Sequenzierung des einzelnen Gens spa, basiert die MLST auf der
Sequenzierung von insgesamt sieben Haushaltsgenen, die verschiedene
Sequenztypen (ST) definieren, welche zu klonalen Komplexen (CC) gruppiert werden.
STs und CCs bilden heutzutage das Gerüst der internationalen SA Nomenklatur in
epidemiologischen Studien. Eine besondere Technik stellen DNA Microarrays (MA)
dar, mit deren Hilfe ST und CC aus charakteristischen Hybridisierungsmustern
zahlreicher Genloci durch Vergleich mit einer Datenbank einer großen
Stammsammlung abgeleitet werden. Neben dieser phylogenetischen Zuordnung
besitzt der DNA MA zahlreiche Loci für Adhäsions-, Virulenz- und Toxingene und liefert
daher eine große Masse an Daten. Der Vergleich zahlreicher, mittels DNA MA
untersuchter, Isolate erfordert daher zur Interpretation und Präsentation ergänzende
bioinformatische Methoden.
Insgesamt gibt es für SA eine umfangreiche Studienlage zur molekulargenetisch
typisierten Infektionsepidemiologie; überwiegend bezieht diese sich jedoch auf Länder
mit einem hohen Standard mikrobieller Diagnostik. Für Afrika gibt es nur wenige
molekular-epidemiologische Daten von SA (inklusive Methicillin-resistenter SA
[MRSA]), insbesondere von nicht nosokomialen (d.h., nicht im Krankenhaus
erworbenen) SA (wie z.B. ambulant erworbener (CA) Methicillin-resistenter oder -
empfindlicher SA [MRSA, MSSA]). Um diese Wissenslücke zu füllen, wurden als
Grundlage dieser Promotionsarbeit vier Studien durchgeführt zur Untersuchung a)
verschiedener bioinformatischer Werkzeuge zur Analyse der molekularen
Epidemiologie von SA, b) der Epidemiologie von afrikanischen und deutschen CAMSSA/
MRSA, die nicht in Gesundheitsinstitutionen erworben wurden, und c) der
Anwendbarkeit der Massenspektrometrie zur Analyse der Epidemiologie von SA.
Zur Charakterisierung der bioinformatischen Werkzeuge wurde das
Subtypisierungspotential der spa-Typisierung und der DNA MA Analyse in einer Studie
mit 46 nasalen CA-MSSA und 46 von Infektionen stammenden CA-MRSA des
Bundeslandes Saarland verglichen. Zudem wurden die Ergebnisse mit drei
bioinformatischen Methoden („splits graph“, hierarchische agglomerative Gruppierung [HAC] und Hauptkomponentenanalyse [PCA]) zur DNA Analyse evaluiert. Um die
Anwendbarkeit der DNA MA Analytik auch in afrikanischen Kohorten zu überprüfen,
wurde eine relativ kleine Isolatkohorte von 52 nigerianischen MSSA Isolaten mittels
DNA MA und splits graph Analyse untersucht. In einer großen, multizentrischen,
afrikanisch-deutschen, geographisch vergleichenden Querschnittsuntersuchung
wurden 1200 CA-SA Isolate, gesunder Freiwilliger und von Patienten aus Sub-Sahara
Afrika und Deutschland, mittels DNA MA analysiert. Zudem wurden Assoziations- und
vergleichende Analysen der CC, ST und der Genausstattung der Isolate, hinsichtlich
ihrer geographischen Herkunft (Deutschland oder Afrika) bzw. ihres
klinischen/kommensalen Ursprung, durchgeführt. Schließlich wurde die mögliche
Anwendbarkeit der Identifikation von CCs oder spa–Typen von CA-SA des CC5, CC8,
CC15, CC30, CC45, CC121 und der spa- Typen t002, t003, t504 basierend auf MALDITOF
Massenspektrometrieprofilen analysiert.
Die mit diesen Untersuchungen angestrebten Ergebnisse, zur Epidemiologie von CAMSSA/
MRSA, wurden zunächst mit der DNA MA Studie des Bundeslandes Saarland
gewonnen, welche ein höheres Subtypisierungspotential für den DNA MA im Vergleich
zur spa–Typisierung zeigte. Die Auswertung der DNA MA Daten mittels der zuvor
genannten drei bioinformatischen Methoden zeigte eine unterschiedliche Zuordnung
von CC5 Isolaten zu 3 bis 7 Isolatgruppen mit Ausnahme von zwölf CC5 Isolaten.
Diese zwölf CC5 Isolate wurden immer zwei identischen Isolatgruppen zugeordnet.
Dies heißt nicht, dass die Methoden fehlerhaft sind, sondern dass die Anwendung
unterschiedlicher mathematischen Algorithmen, die entweder die Unterschiede oder
die Ähnlichkeiten der Hybridisierungsmuster untersuchen, zu geringfügigen
Unterschieden führen. Zudem zeigten die MSSA eine hohe Diversität im Vergleich zur
CA-MRSA Gruppe, die vom CC5 Rhine-Hesse epidemischen Stamm dominiert wurde
(89%).
Die weitere Analyse afrikanischer Stämme aus der Studie mit nigerianischen MSSA
zeigte eine heterogene und divergente Natur der untersuchten nigerianischen MSSA
und bestätigte, dass der DNA MA eine geeignete Methode zur Genotypisierung
afrikanischer SA ist. Die Ergebnisse der multizentrischen deutsch-afrikanischen
Konsortialstudie wies innerhalb der Isolatgruppe insgesamt 40 verschiedene CCs und
STs nach, einschließlich 5 neuer STs. Die Mehrheit, 15 von 22 (68%) der CCs, die
auch am häufigsten identifiziert wurden, waren entweder in Sub-Sahara Afrika (Gabon,
Mozambique, Tanzania) (CC80, CC88, CC121, CC152) oder Deutschland (CC22, CC30, CC45, CC398) vorherrschend. Die Rate an MRSA Trägern in der afrikanischen
(3%) und deutschen Bevölkerung (1%) war sehr gering, vermutlich durch die auf CASA
beschränkte Untersuchung. Die Analyse der Genprofile zeigte eine hohe Prävalenz
der Panton-Valentin Leukozidin kodierenden Gene lukF/S-PV in afrikanischen
Isolaten. Allgemein konnten kontinentspezifische Unterschiede für die CC und CA-SA
Genprofile identifiziert werden. Zudem wurde ersichtlich, dass ergänzend zur guten
Erstcharakterisierung durch den DNA MA, standardisierte, komplexe analytische
Methoden für Assoziationsanalysen und epidemiologische Untersuchungen
erforderlich sind.
Das letzte Untersuchungsziel, die Bewertung vergleichender
Massenspektrometrieanalysen zeigte, dass - basierend auf ihrem Spektrometrieprofil
- nur SA des CC121 korrekt identifiziert werden konnten, während keine korrekte
Identifikation anderer CC oder spa-Typen möglich war.
Zusammenfassend identifizierte diese Arbeit länderspezifische Unterschiede in der
Verteilung klonaler Komplexe und spezifischer Gene in den untersuchten Regionen,
insbesondere bei alleiniger Betrachtung klinischer Isolate. Sie gibt wertvolle Hinweise
auf die genetisch basierten Gründe, welche die angeblichen Unterschiede in der
klinischen Präsentation und dem Verlauf von durch SA verursachten Infektionen in sich
entwickelnden und industriellen Ländern erklären könnten. Zudem wurde gezeigt, dass
der DNA MA im Gegensatz zur Massenspektrometrie eine geeignete Methode zur SA
Charakterisierung z.B. in Ausbruchssituationen ist. Es darf jedoch nicht unerwähnt
bleiben, dass als besondere Anforderung an eine anspruchsvolle, DNA MA-basierte
phylogenetische Analytik mehrerer Isolate die Anwendung komplexer
bioinformatischer Analysemethoden zur Interpretation erforderlich ist, und dass die
Charakteristiken und Algorithmen der gewählten bioinformatischen Methode zu
geringfügigen Unterschieden in der Dateninterpretation führen kann
Recommended from our members
Genomic surveillance of methicillin-resistant Staphylococcus aureus at local, regional and national levels
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infection, which is associated with increased cost and length of hospital stays and substantial morbidity and mortality. The rapid progression of whole-genome sequencing (WGS) technologies over the past decade has provided highly detailed and discriminatory insights into the epidemiology of MRSA. However, WGS is still not implemented in the routine surveillance of MRSA at a local, regional or national level. This thesis applies WGS to a number of questions and populations to describe its benefits.
Standard infection control investigation commonly uses the antimicrobial susceptibility patterns (ASPs, patterns of susceptibility and resistance to commonly used antibiotics) of MRSA as a surrogate for bacterial relatedness. As they are readily available, ASPs are often combined with patient movement to evaluate putative MRSA outbreaks in hospitals. The accuracy of this method was evaluated by comparing linked cases based on MRSA ASPs versus linked cases based on whole-genome relatedness, using data from a year-long prospective observational cohort study of 1,465 MRSA-positive individuals. The sensitivity and specificity of ASP in the presence of a direct ward contact was 44% and 85%, respectively; in the presence of a shared residential post code, the sensitivity and specificity of ASP is 59% and 76%, respectively. This demonstrates that compared to WGS plus epidemiology, ASP and epidemiology does not reliably identify or refute transmission events.
A lineage referred to as epidemic (E)-MRSA15 is largely considered to be associated with hospital settings, but an epidemiological and genomic investigation of a MRSA outbreak in a General Practice (GP) identified 15 people who were E-MRSA15 positive, the majority of which shared a link to a leg ulcer/podiatry clinic in the GP surgery. The outbreak had not been detected previously, and was only identified post-hoc from MRSA sequence data, highlighting the importance of MRSA sequencing to detect otherwise cryptic community outbreaks.
The utility of WGS for the investigation of regional MRSA epidemiology was explored for two potentially high-risk MRSA lineages (USA300 and ST2371) that are otherwise unmonitored by current surveillance. Screening the genomes of MRSA isolated from 1,465 people identified over a 12-month period demonstrated that 4.2% of cases were positive for MRSA USA300, with multiple introductions and household transmissions identified. Five people were positive for ST2371, all of whom had a direct or indirect link to a substantial outbreak in an intensive care unit at Addenbrooke’s Hospital in 2011, thus confirming the value of WGS in regional epidemiological investigations.
The feasibility and utility of incorporating WGS into routine national MRSA surveillance was evaluated through a combined epidemiological and genomic survey of MRSA bacteraemia undertaken in England over a one-year period. This captured 903 reported cases of MRSA bacteraemia, with 425 isolates available for sequencing. Almost two thirds of isolates were assigned to multi-locus sequence type clonal complex (CC) 22. The addition of MRSA genomes from published outbreak investigations showed that the study genomes could provide context for outbreak isolates and supported cluster identification. Potentially high-risk lineages were also detected. These findings support the integration of epidemiological and genomic surveillance for MRSA bacteraemia as a first step towards a comprehensive national surveillance programme
- …