Isolierung und Typisierung von Salmonellen aus Trinkwasserquellen in Benin, Westafrika

Die Situation der Trinkwasserversorgung im ländlichen oberen Ouémé Einzugsgebiet in Benin, Westafrika, ist dadurch gekennzeichnet, dass viele Menschen keinen Zugang zu sicherem Trinkwasser haben. Am häufigsten stehen offene traditionelle und moderne Brunnen, die 90 % aller Wasserquellen ausmachen, zur Trinkwasserversorgung zur Verfügung. Bohrlöcher mit geschlossenen Pumpenanlagen, die bakteriologisch unbedenkliches Wasser liefern, machen nur 6 % aller Wasserstellen aus. Aufgrund unzureichender Trinkwasserhygiene und eines Mangels an grundlegenden sanitären Einrichtungen sind 70 % aller Wasserquellen mit dem Fäkalindikator Escherichia coli kontaminiert. Für diese Arbeit wurde Salmonella enterica neben E. coli als Indikatorkeim zur Einschätzung der Infektionsgefahr für die Wasserkonsumenten nachgewiesen. In 8 % der untersuchten Wasserstellen konnten Kontaminationen durch enteritische Salmonellen detektiert werden. In dem Wasser moderner Brunnen, die aufgrund ihrer baulichen Eigenschaften vor dem Eintrag von Kontaminationen in das Wasser geschützter sind als traditionelle Brunnen, konnte ebenfalls in 17 Fällen enteritische Salmonellen nachgewiesen werden. Die Stämme konnten vielen verschiedenen, selten vorkommenden Serotypen zugeordnet werden, von denen keiner vorherrschend in der Region vorkam. Insgesamt gehörten 177 Salmonella-Stämme zu 79 verschiedenen Serotypen. Ein bislang unbekannter Serotyp mit der Antigenformel wurde als S. Parakou in das White-Kauffmann-Le Minor Schema übernommen. Untersuchungen der Stämme mittels Multilocus Sequence Typing (MLST) ergaben, dass es sich um eine Vielzahl bislang unbekannter Sequenztypen (ST) handelt, die in die S.enterica MLST-Datenbank eingefügt wurden. Zur Einschätzung des Infektionsrisikos für die Wasserkonsumenten wurden die als Salmonella outer proteins (Sop) bezeichneten Virulenz-assoziierten Effektorproteine SopB. SopD, SopE1, SopE2 und AvrA auf DNA- und Protein-Ebene für die Umwelt-Isolate nachgewiesen. 66 % der Stämme zeigten ein Expressionsmuster, das dem entspricht, das typischerweise für klinische Enteritis-Isolate nachgewiesen wurde. Untersuchungen von Stuhlproben der Einwohner eines Dorfes, in dem verschiedene Wasserstellen durch Salmonellen kontaminiert waren, haben ergeben, dass bei 6,5 % der Probanden der Sero � und Sequenztyp aus der Stuhlprobe mit dem der genutzten Wasserquelle übereinstimmte

Genomic diversity of Salmonella enterica -The UoWUCC 10K genomes project

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase , which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing

Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents

Detection of Adenoviruses and Rotaviruses in Drinking Water Sources Used In Rural Areas of Benin, West Africa▿

Diseases associated with viruses also found in environmental samples cause major health problems in developing countries. Little is known about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. We established a method to analyze 10 liters of water from drinking water sources in a rural area of Benin for the presence of adenoviruses and rotaviruses. Overall, 541 samples from 287 drinking water sources were tested. A total of 12.9% of the sources were positive for adenoviruses and 2.1% of the sources were positive for rotaviruses at least once. Due to the temporary nature of viral contamination in drinking water sources, the probability of virus detection increased with the number of samples taken at one test site over time. No seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Overall, 3 of 15 surface water samples (20%) and 35 of 247 wells (14.2%) but also 2 of 25 pumps (8%) tested positive for adenoviruses or rotaviruses. The presence of latrines within a radius of 50 m in the vicinity of pumps or wells was identified as being a risk factor for virus detection. In summary, viral contamination was correlated with the presence of latrines in the vicinity of drinking water sources, indicating the importance of appropriate decision support systems in these socioeconomic prospering regions

Correction: Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica.

[This corrects the article DOI: 10.1371/journal.ppat.1002776.]

Comparison of groupings according to eBGs <i>versus</i> groupings by other algorithms.

<p>Note: Bootstrap is an abbreviation for the Gene by Gene Bootstrap approach with 50% support. 1092 distinct STs were tested by all three methods. BAPS with an upper bound of 400 assigned all STs to 216 clusters. The two other methods identified singletons (ClonalFrame, 189) or excluded individual STs whose branches did not receive 50% support (Bootstrap, 569), which were excluded from further comparisons. No. eBGs per cluster shows the numbers of clusters that contained 0, 1, 2,…7. eBGs according to each of the three methods. Clusters per eBG indicates the number of clusters identified by each of the three methods to which any STs within an eBG were assigned. Maximal number of clusters per eBG: BAPS, 2; ClonalFrame, 2; Bootstrap, 4. The significance of these associations was tested by 10,000 permutations of assignments of STs to eBGs for each of the three clustering assignments or by 10,000 permutations of the clustering assignments for the real eBG assignments of STs. None of the 10,000 permutations exceeded the number of eBGs found per cluster or the number of eBGs assigned to one cluster except that 9.5% of the permutations of the number of eBGs per BAPs cluster equalled or exceeded 71.</p

Variant amino acids in the FljB protein.

<p>Sequences of FljB (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002776#ppat.1002776.s016" target="_blank">Table S8</a>) from isolates investigated here and additional sequences from GenBank with ≥95% BLAST identity and 100% coverage were trimmed to a length of 440 amino acids, beginning at amino acid 36 in the FljB protein from strain LT-2. The figure shows all differences relative to the uppermost sequence (strain SL3261). FljB protein groups were assigned with the help of a UPGMA tree (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002776#ppat.1002776.s007" target="_blank">Fig. S7</a>) and are indicated at the right, together with the serological factors in the phase 2 flagellar antigen. Strain and serovar designations are at the left, followed by MLST ST and eBG designations for the strains investigated here.</p

Genomic diversity of Salmonella enterica - The UoWUCC 10K genomes project

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase, which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing

MSTree of 6,7:c:1,5 isolates.

<p>Details are as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002776#ppat-1002776-g004" target="_blank">Fig. 4</a> and additional information can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002776#ppat-1002776-t003" target="_blank">Tables 3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002776#ppat.1002776.s015" target="_blank">S7</a>.</p

Biotypes associated with serovars within 6,7:c:1,5 <i>S. enterica</i>.

*<p>d- tartrate fermentation is only used to identify Typhisuis.</p>‡<p>Decatur was previously referred to as Choleraesuis var. Decatur <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002776#ppat.1002776-LeMinor1" target="_blank">[65]</a>.</p>§<p>Dulcitol is fermented by Decatur in eBGs 141 and 144 and STs 70 and 637 but not by eBG142.</p><p>The nutritional correlations between the different serovars are based on published information <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002776#ppat.1002776-Grimont1" target="_blank">[2]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002776#ppat.1002776-LeMinor1" target="_blank">[65]</a> after modification due to the experiments described here.</p