Microbiological survey and genomic analysis of Cronobacter sakazakii strains isolated from US households and retail foods.

Cronobacter species are opportunistic pathogens that are capable of causing morbidity and mortality, particularly in infants. Although the transmission dynamics involved in Cronobacter infections remain largely unknown, contaminated powdered infant formula (PIF) has been linked to 30% of Cronobacter sakazakii cases involving invasive illness in infants. As several lines of evidence have implicated the domestic environment in PIF contamination, we undertook a microbiological survey of homes (N = 263) across the US. Cronobacter spp. and C. sakazakii were isolated from 36.1% and 24.7% of US homes, respectively, with higher recovery rates observed for floor and kitchen surfaces. Multi-locus sequence typing indicated that the dominant strain was C. sakazakii ST4, the sequence type most commonly associated with neonatal meningitis. For comparison purposes, retail foods (N = 4,009) were also surveyed, with the highest contamination frequencies (10.1%-26.3%) seen for nut products, seeds, and grains/baked goods/flours. The sequence type profile of isolates recovered from homes mirrored that of isolates recovered from retail foods, with increased representation of ST1, ST4, ST13, ST17, and ST40. Analysis of 386 whole genomic sequences revealed significant diversity. Redundancies were only observed for isolates recovered from within the same domicile, and there were no identical matches with sequences archived at the NCBI pathogen database. Genes coding for putative virulence and antibiotic resistance factors did not segregate with clinically significant sequence types. Collectively, these findings support the possibility that contamination events occurring within the home should not be overlooked as a contributor to community-onset Cronobacter infections. IMPORTANCE: Cronobacter sakazakii is an opportunistic pathogen that can cause significant morbidity and mortality in neonates. Its transmission dynamics are poorly understood, though powered infant formula (PIF) is thought to be the major transmission vehicle. How the PIF becomes contaminated remains unknown. Our survey shows that roughly 1/4 of US homes are contaminated with Cronobacter sakazakii, particularly in the kitchen setting. Our analyses suggest that the domestic environment may contribute to contamination of PIF and provides insights into mitigating the risk of transmission.

Acquisition of the L452R Mutation in the ACE2-Binding Interface of Spike Protein Triggers Recent Massive Expansion of SARS-CoV-2 Variants.

We report that there is a recent global expansion of numerous independent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with mutation L452R in the receptor-binding domain (RBD) of the spike protein. The massive emergence of L452R variants was first linked to lineage B.1.427/B.1.429 (clade 21C) that has been spreading in California since November and December 2020, originally named CAL.20C and currently variant of interest epsilon. By PCR amplification and Sanger sequencing of a 541-base fragment coding for amino acids 414 to 583 of the RBD from a collection of clinical specimens, we identified a separate L452R variant that also recently emerged in California but derives from the lineage B.1.232, clade 20A (named CAL.20A). Notably, CAL.20A caused an infection in gorillas in the San Diego Zoo, reported in January 2021. Unlike the epsilon variant that carries two additional mutations in the N-terminal domain of spike protein, L452R is the only mutation found in the spike proteins of CAL.20A. Based on genome-wide phylogenetic analysis, emergence of both viral variants was specifically triggered by acquisition of L452R, suggesting a strong positive selection for this mutation. Global analysis revealed that L452R is nearly omnipresent in a dozen independently emerged lineages, including the most recent variants of concern/interest delta, kappa, epsilon and iota, with the lambda variant carrying L452Q. L452 is in immediate proximity to the angiotensin-converting enzyme 2 (ACE2) interaction interface of RBD. It was reported that the L452R mutation is associated with immune escape and could result in a stronger cell attachment of the virus, with both factors likely increasing viral transmissibility, infectivity, and pathogenicity.

Unification of Abyssicoccus albus Jiang et al. 2016 and Auricoccus indicus Prakash et al. 2017 and the status of the genus Auricoccus Prakash et al. 2017.

Based on the high phylogenetic relatedness of Auricoccus indicus Prakash et al. 2017 and Abyssicoccus albus Jiang et al. 2016, it is proposed to unite them with retaining the latter name as having nomenclatural priority. As the result of the species unification, the genus Auricoccus name is proposed to consider as illegitimate in the boundaries determined by Rule 51a of the International Code of Nomenclature of Prokaryotes.

Reclassification of Burkholderia insecticola as Caballeronia insecticola comb. nov. and reliability of conserved signature indels as molecular synapomorphies.

In the last 4 years, most of the species previously classified as members of the genus Burkholderia have been transferred to the novel genera Paraburkholderia, Caballeronia, Robbsia, Mycetohabitans and Trinickia. However, there have been objections to splitting the genus Burkholderiasensu lato, and based on this taxonomic opinion, strain RPE64T, which has the 16S rRNA gene sequence identical to that of Caballeronia peredens LMG 29314T, has recently been proposed as the type strain of Burkholderia insecticolasp. nov. The arguments against the split were analysed in this study and found to be not convincing enough to revise the taxonomic positions of members of the novel genera. Therefore, based on the results of phylogenetic analyses, including comparisons of 16S rRNA gene sequences and those of concatenated proteins, as well as on the fact that strain RPE64T had all molecular signatures included as Caballeronia-specific markers in the genus description, we propose to reclassify B. insecticola as Caballeronia insecticola comb. nov. The results of this study also showed that 'Burkholderia novacaledonica' and 'Burkholderia ultramafica' should be transferred to the genera Caballeronia and Paraburkholderia, respectively.

Reclassification of Eubacterium combesii and discrepancies in the nomenclature of botulinum neurotoxin-producing clostridia: Challenging Opinion 69. Request for an Opinion.

To clarify the taxonomic position of Eubacterium combesii, the whole genome of its type strain, DSM 20696T, was sequenced. Comparison of this sequence with known sequences of other bacteria confirmed that E. combesii represented a member of the Clostridium sporogenes/Clostridium botulinum Group I clade. However, the results of phylogenetic analysis also demonstrated that the latter two species did not form the same genetic entity and that E. combesii was in the C. botulinum Group I subclade. Meanwhile, we showed that E. combesii DSM 20696T did not produce botulinum neurotoxins (BoNTs) and thus should be identified as a strain of C. sporogenes in accordance with the current nomenclature of BoNT-producing clostridia, which is based, in particular, on Opinion 69 issued by the Judicial Commission of the ICSB. However, review of the corresponding Request for an Opinion revealed that it had been based on an erroneous statement. Therefore, we request reconsideration of Opinion 69 and propose to reclassify Eubacterium combesii as a later synonym of Clostridium botulinum. The results of phylogenetic analysis of the other five groups of BoNT-producing clostridia indicated that all the groups were far distant from each other. However, the members of Groups IV-VI are classified as strains of different species, while all members of Groups I-III are designated C. botulinum. Meanwhile, similarly to Group I, Groups II and III are also polyphyletic and appear to consist of two and four species, respectively. These results demonstrate, once again, discrepancies in the nomenclature of BoNT-producing bacteria and corroborate our request for reconsideration of Opinion 69.

Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay.

A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.

Transfer of 13 species of the genus Burkholderia to the genus Caballeronia and reclassification of Burkholderia jirisanensis as Paraburkholderia jirisanensis comb. nov.

A recent study of a group of Burkholderia glathei-like bacteria resulted in the description of 13 novel species of the genus Burkholderia. However, our analysis of phylogenetic positions of these species and their molecular signatures (conserved protein sequence indels) showed that they belong to the genus Caballeronia, and we propose to transfer them to this genus. The reclassified species names are proposed as Caballeroniaarationis comb. nov., Caballeroniaarvi comb. nov., Caballeroniacalidae comb. nov., Caballeroniacatudaia comb. nov., Caballeroniaconcitans comb. nov., Caballeroniafortuita comb. nov., Caballeroniaglebae comb. nov., Caballeroniahypogeia comb. nov., Caballeroniapedi comb. nov., Caballeroniaperedens comb. nov., Caballeroniaptereochthonis comb. nov., Caballeroniatemeraria comb. nov. and Caballeronia turbans comb. nov. It is also proposed to reclassify Burkholderia jirisanensis as Paraburkholderiajirisanensis comb. nov. Based on the results of the polyphasic study, B. jirisanensis had been described as a member of the A-group of the genus Burkholderiaand the most closely related to Burkholderia rhizosphaerae, Burkholderia humisilvae and Burkholderia solisilvae currently classified as belonging to the genus Paraburkholderia.

Clostridium tepidum sp. nov., a close relative of Clostridium sporogenes and Clostridium botulinum Group I.

Obligately anaerobic, Gram-stain-positive, spore-forming bacteria indistinguishable by pulsed-field gel electrophoresis were isolated from non-dairy protein shakes in bloated bottles. One of the isolates, strain IEH 97212T, was selected for further study. The strain was closely related to Clostridium sporogenes and Clostridium botulinum Group 1 based on 16S rRNA gene sequence similarities. Phylogenetic analysis also showed that strain IEH 97212T and strain PE (=DSM 18688), a bacterium isolated from solfataric mud, had identical 16S rRNA gene sequences. Strains IEH 97 212T and DSM 18 688 were relatively more thermophilic (temperature range for growth: 30-55 °C) and less halotolerant [growth range: 0-2.5 % (w/v) NaCl] than C. sporogenes and C. botulinum. They were negative for catalase, oxidase, urease and l-pyrrolidonyl-arylamidase and did not produce indole. The strains produced acid from d-glucose, maltose and trehalose, and hydrolysed gelatin, but did not hydrolyse aesculin. The end-products of growth included acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, isocaproic acid, phenylpropionic acid, 2-piperidinone, 2-pyrrolidinone and gas(es). The predominant fatty acids were C14 : 0, C16 : 0 and C18 : 1ω9c. The genomic DNA G+C content of strains IEH 97212T and DSM 18688 was 26.9 and 26.7 mol%, respectively. According to the digital DNA-DNA hybridization data, the relatedness of these strains was 98.4 %, while they showed only 35.7-36.0 % relatedness to C. sporogenes. Based on the results of this polyphasic study, these strains represent a novel species, for which the name Clostridium tepidum sp. nov. is proposed, with the type strain IEH 97212T (=NRRL B-65463T=DSM 104389T).

Transfer of eleven species of the genus Burkholderia to the genus Paraburkholderia and proposal of Caballeronia gen. nov. to accommodate twelve species of the genera Burkholderia and Paraburkholderia.

It has been proposed to split the genus Burkholderia into two genera according to phylogenetic clustering: (1) a genus retaining this name and consisting mainly of animal and plant pathogens and (2) the genus Paraburkholderia including so-called environmental bacteria. The latter genus name has been validly published recently. During the period between the effective and valid publications of the genus name Paraburkholderia, 16 novel species of the genus Burkholderiawere described, but only two of them can be classified as members of this genus based on the emended genus description. Analysis of traits and phylogenetic positions of the other 11 species shows that they belong to the genus Paraburkholderia, and we propose to transfer them to this genus. The reclassified species names are proposed as Paraburkholderia dipogonis comb. nov., Paraburkholderia ginsengiterrae comb. nov., Paraburkholderia humisilvae comb. nov., Paraburkholderia insulsa comb. nov., Paraburkholderia kirstenboschensis comb. nov., Paraburkholderia metalliresistens comb. nov., Paraburkholderia monticola comb. nov., Paraburkholderia panaciterrae comb. nov., Paraburkholderia rhizosphaerae comb. nov., Paraburkholderia solisilvae comb. nov. and Paraburkholderia susongensis comb. nov. The remaining three species are transferred to the new genus Caballeronia gen. nov. proposed to accommodate twelve species of the genera Burkholderia and Paraburkholderia forming a distinctive clade in phylogenetic trees. The new genus members are Caballeronia choica comb. nov., Caballeronia cordobensis comb. nov., Caballeronia glathei comb. nov., Caballeronia grimmiae comb. nov., Caballeronia humi comb. nov., Caballeronia megalochromosomata comb. nov., Caballeronia jiangsuensis comb. nov., Caballeronia sordidicola comb. nov., Caballeronia telluris comb. nov., Caballeronia terrestris comb. nov., Caballeronia udeis comb. nov., and Caballeronia zhejiangensis comb. nov.

The status of the genus Pseudoroseovarius Sun et al. 2015.

The results of phylogenetic analyses of the genera Aliiroseovarius Park et al. 2015 and Pseudoroseovarius Sun et al. 2015 and comparison of the phenotypic features of their members showed that these genera should be united. Based on nomenclatural priority of the genus Aliiroseovarius, it is proposed to reclassify Pseudoroseovarius crassostreae as a later homotypic synonym of Aliiroseovarius crassostreae, Pseudoroseovarius halocynthiae as a later homotypic synonym of Aliiroseovarius halocynthiae, Pseudoroseovarius sediminilitoris as a later homotypic synonym of Aliiroseovarius sediminilitoris and Pseudoroseovarius zhejiangensis as Aliiroseovarius zhejiangensis comb. nov.

Reclassification of Paraburkholderia panaciterrae (Farh et al. 2015) Dobritsa & Samadpour 2016 as a later synonym of Paraburkholderia ginsengiterrae (Farh et al. 2015) Dobritsa & Samadpour 2016.

Whole-genome sequencing and PFGE analysis of Paraburkholderia ginsengiterrae DCY85T and Paraburkholderia panaciterrae DCY85-1T showed these strains are highly similar and may even be clones of the same strain. The PFGE patterns of XbaI-, AvaII-, and SpeI-digested genomic DNA of the two strains were indistinguishable. Based on the priority of valid publications of the species basonyms, Burkholderia ginsengiterrae and Burkholderia panaciterrae, it is proposed to reclassify P. panaciterrae as a later synonym of P. ginsengiterrae. The P. ginsengiterrae description was emended by replacing the DNA G+C content value of 66.0 mol%, which is higher than the 65 mol% considered the threshold for species of the genus Paraburkholderia, with the value of 62.4-62.5 mol%, calculated as the mean DNA G+C content of the draft genomes of strains DCY85-1T and DCY85T.

Identification of strains Bacillus aerophilus MTCC 7304T as Bacillus altitudinis and Bacillus stratosphericus MTCC 7305T as a Proteus sp. and the status of the species Bacillus aeriusShivaji et al. 2006. Request for an Opinion.

On the basis of 16S rRNA, rpoB, gyrB and pycA gene sequence analyses, characterization of biochemical features and other phenotypic traits and pulsed-field gel electrophoresis (PFGE) fingerprinting, it was ascertained that strains Bacillus aerius MTCC 7303T, Bacillus aerophilus MTCC 7304(T) and Bacillus stratosphericus MTCC 7305(T) do not conform to the descriptions of the type strains of the respective species. Strains MTCC 7303(T) and MTCC 7304(T) were indistinguishable from Bacillus altitudinis DSM 21631(T), while strain MTCC 7305(T) should be classified as a representative of a Proteus sp. Our attempts to find other deposits of the type strains of these species were unsuccessful. Therefore, the results support the Request for an Opinion on the status of the species Bacillus aerophilus and Bacillus stratosphericus by Branquinho et al. [Branquinho, R., Klein, G., Kämpfer, P. & Peixe, L. V. (2015). Int J Syst Evol Microbiol 65, 1101]. It is also proposed that the Judicial Commission should place the name Bacillus aerius on the list of rejected names if a suitable replacement type strain cannot be found or a neotype is not proposed within two years following the publication of this Request (Rule 18c).

Evolution of Salmonella enterica virulence via point mutations in the fimbrial adhesin.

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella.

Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray).

This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

Reprint of "Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray)".

This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

Escherichia coli O157:H7 infections associated with consumption of locally grown strawberries contaminated by deer.

BACKGROUND: An outbreak of Escherichia coli O157:H7 was identified in Oregon through an increase in Shiga toxin-producing E. coli cases with an indistinguishable, novel pulsed-field gel electrophoresis (PFGE) subtyping pattern. METHODS: We defined confirmed cases as persons from whom E. coli O157:H7 with the outbreak PFGE pattern was cultured during July-August 2011, and presumptive cases as persons having a household relationship with a case testing positive for E. coli O157:H7 and coincident diarrheal illness. We conducted an investigation that included structured hypothesis-generating interviews, a matched case-control study, and environmental and traceback investigations. RESULTS: We identified 15 cases. Six cases were hospitalized, including 4 with hemolytic uremic syndrome (HUS). Two cases with HUS died. Illness was significantly associated with strawberry consumption from roadside stands or farmers' markets (matched odds ratio, 19.6; 95% confidence interval, 2.9-∞). A single farm was identified as the source of contaminated strawberries. Ten of 111 (9%) initial environmental samples from farm A were positive for E. coli O157:H7. All samples testing positive for E. coli O157:H7 contained deer feces, and 5 tested farm fields had ≥ 1 sample positive with the outbreak PFGE pattern. CONCLUSIONS: The investigation identified fresh strawberries as a novel vehicle for E. coli O157:H7 infection, implicated deer feces as the source of contamination, and highlights problems concerning produce contamination by wildlife and regulatory exemptions for locally grown produce. A comprehensive hypothesis-generating questionnaire enabled rapid identification of the implicated product. Good agricultural practices are key barriers to wildlife fecal contamination of produce.

Genetic markers for rapid PCR-based identification of gull, Canada goose, duck, and chicken fecal contamination in water.

Avian feces contaminate waterways but contribute fewer human pathogens than human sources. Rapid identification and quantification of avian contamination would therefore be useful to prevent overestimation of human health risk. We used subtractive hybridization of PCR-amplified gull fecal 16S RNA genes to identify avian-specific fecal rRNA gene sequences. The subtracters were rRNA genes amplified from human, dog, cat, cow, and pig feces. Recovered sequences were related to Enterobacteriaceae (47%), Helicobacter (26%), Catellicoccus (11%), Fusobacterium (11%), and Campylobacter (5%). Three PCR assays, designated GFB, GFC, and GFD, were based on recovered sequence fragments. Quantitative PCR assays for GFC and GFD were developed using SYBR green. GFC detected down to 0.1 mg gull feces/100 ml (corresponding to 2 gull enterococci most probable number [MPN]/100 ml). GFD detected down to 0.1 mg chicken feces/100 ml (corresponding to 13 Escherichia coli MPN/100 ml). GFB and GFC were 97% and 94% specific to gulls, respectively. GFC cross-reacted with 35% of sheep samples but occurred at about 100,000 times lower concentrations in sheep. GFD was 100% avian specific and occurred in gulls, geese, chickens, and ducks. In the United States, Canada, and New Zealand, the three markers differed in their geographic distributions but were found across the range tested. These assays detected four important bird groups contributing to fecal contamination of waterways: gulls, geese, ducks, and chickens. Marker distributions across North America and in New Zealand suggest that they will have broad applicability in other parts of the world as well.

Escherichia coli O157:H7 Cluster Associated With Deer Harvested at a Single Wildlife Hunting Area, Oregon, 2017.

The Oregon Health Authority routinely investigates clusters of reportable enteric diseases identified by whole-genome sequencing. While investigating 2 cases of Escherichia coli O157:H7 in 2019, in which both patients were exposed to the same home-processed "jerky" and clinical isolates matched within 2 single nucleotide polymorphisms (SNPs), we discovered, by searching the National Library of Medicine's National Center for Biotechnology Information website, 3 other cases of E coli O157:H7 from 3 Oregon counties-Tillamook, Umatilla, and Douglas-whose clinical isolates were within 9 SNPs of the 2 initial matched cases. We analyzed interview data for 3 case patients and followed up with additional hypothesis-generating questions. Onset of illness for the Tillamook, Umatilla, and Douglas county cases were October 7, 2017, October 27, 2017, and April 30, 2018, respectively. The median age of the 5 case patients was 16 years. Parents of 2 of the 5 case patients, each from a different county, had harvested deer approximately 20 miles from each other in the same Douglas County wildlife hunting unit in late September 2017. The case from Umatilla County was lost to follow-up. Although it is well documented that deer are a viable and substantial reservoir of E coli O157:H7, to our knowledge, this is the first time that venison from a common wildlife hunting unit was found to be associated with a cluster of illnesses. This finding suggests a geographic nidus for E coli O157:H7. We recommend routinely asking about wildlife hunting units when developing exposure hypotheses involving potential venison-associated clusters.