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. 2019 May 2;85(10):e00139-19.
doi: 10.1128/AEM.00139-19. Print 2019 May 15.

Clinically Unreported Salmonellosis Outbreak Detected via Comparative Genomic Analysis of Municipal Wastewater Salmonella Isolates

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Clinically Unreported Salmonellosis Outbreak Detected via Comparative Genomic Analysis of Municipal Wastewater Salmonella Isolates

Sabrina Diemert et al. Appl Environ Microbiol. .
Free PMC article

Abstract

Municipal wastewater includes human waste that contains both commensal and pathogenic enteric microorganisms, and this collective community microbiome can be monitored for community diseases. In a previous study, we assessed the salmonellosis disease burden using municipal wastewater from Honolulu, Hawaii, which was monitored over a 54-week period. During that time, a strain of Salmonella enterica serovar Paratyphi B variant L(+) tartrate(+) (also known as Salmonella enterica serovar Paratyphi B variant Java) was identified; this strain was detected simultaneously with a clinically reported outbreak, and pulsed-field gel electrophoresis patterns were identical for clinical and municipal wastewater isolates. Months after the outbreak subsided, the same pulsotype was detected as the dominant pulsotype in municipal wastewater samples, with no corresponding clinical cases reported. Using genomic characterization (including core single-nucleotide polymorphism alignment, core genome multilocus sequence typing, and screening for virulence and antibiotic resistance genes), all S Java municipal wastewater isolates were determined to be clonal, indicating a resurgence of the original outbreak strain. This demonstrates the feasibility and utility of municipal wastewater surveillance for determining enteric disease outbreaks that may be missed by traditional clinical surveillance methods.IMPORTANCE Underdetection of microbial infectious disease outbreaks in human communities carries enormous health costs and is an ongoing problem in public health monitoring (which relies almost exclusively on data from health clinics). Surveillance of municipal wastewater for community-level monitoring of infectious disease burdens has the potential to fill this information gap, due to its easy access to the mixed community microbiome. In the present study, the genomes of 21 S Java isolates (collected from municipal wastewater in Honolulu) were analyzed; results showed that the same Salmonella strain that caused a known salmonellosis clinical outbreak in spring 2010 remerged as the most dominant strain in municipal wastewater in spring 2011, indicating a new outbreak that was not detected by health clinics. Our results show that wastewater monitoring holds great promise to inform the field of public health regarding outbreak status within communities.

Keywords: comparative genomics; outbreaks; salmonellosis; wastewater.

Figures

FIG 1
FIG 1
Relative abundance of Salmonella serovars in municipal wastewater (MW). Serovar identification was conducted using PFGE. The S. Java clinical frequency distribution (epidemic curve) is underlaid. Asterisks indicate S. Java relative abundances that were significantly greater than the average weekly relative abundance of the most dominant serovar, as determined via Z-test (α values of <0.05, adjusted via the Bonferroni correction for multiple comparisons). Correspondence with the clinically reported outbreak can be seen in 2010 (weeks 1 to 5), with no clinical detection during the detection period in 2011 (weeks 47 to 53).
FIG 2
FIG 2
Phylogenetic tree based on core SNP alignment. The tree is not to scale regarding genetic distance but is illustrative of the topology. The reference was S. Java strain CFSAN024725 (assembly NCBI RefSeq accession no. GCF_000806525.1), and isolates starting with SRR correspond to U.S. clinical samples. Bootstrap support is displayed under the branches, with nucleotide substitutions per site shown above the branches. All municipal water isolates show low phylogenetic distance values under strong bootstrap support (>85%).
FIG 3
FIG 3
Core SNP distance heatmap (values obtained via snp-dists and visualized via Seaborn in Python). Municipal water isolates and 1 clinical isolate (GenBank accession no. SRR7639130, USA-Unknown) have low core SNP differences (0 to 4 SNPs), indicating clonality.
FIG 4
FIG 4
MST for publicly available S. Paratyphi B isolates. The MST was constructed in GrapeTree using the EnteroBase cgMLST v2 method (3,002 loci) (70, 72), using all isolates in EnteroBase databases identified as S. Paratyphi B serovar using the SISTR method (71). White nodes indicate that no relevant metadata (“country” label) were available for the specific sample. Yellow color was added manually to separate Hawaii municipal water samples from the other U.S. samples. The MST is displayed using log-scale collapsed branches for allele distances of <20, with the inset expanded to show all branches (allele distances of ≥1) for the Hawaii municipal water cluster (n = 11 for Hawaii municipal water samples). Gray labels in inset indicate allele distances between nodes. Hawaii municipal water samples form a distinct cluster, with isolates from both sampling years (2010 and 2011) forming a core genotype. Interactive figure available online at https://enterobase.warwick.ac.uk/ms_tree/23518.

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