Received: 14 May 2019 | Revised: 14 August 2019 | Accepted: 15 August 2019 DOI: 10.1111/zph.12647 ORIGINAL ARTICLE An investigation of Salmonella Fluntern illnesses linked to leopard geckos—United States, 2018 Lia Koski1,2 | Emilio DeBess3 | Hilary E. Rosen4 | Roshan Reporter5 | Thomas Waltz1 | Molly Leeper1 | Jeniffer Concepcion Acevedo1 | Renáta Karpíšková6 | Jacquelyn McCormick7 | Tereza Gelbicova6 | Brenda Morningstar-Shaw8 | Megin Nichols1 | Richard F. Leman3 1 Division of Foodborne, Waterborne and Environmental Disease, Centers for Disease Control and Prevention, Atlanta, GA, USA 2 CAITTA, Inc., Herndon, VA, USA 3 Oregon Public Health Division, Portland, OR, USA 4 California Department of Public Health, Sacramento, CA, USA 5 Los Angeles County Department of Public Health, Los Angeles, CA, USA 6 Veterinary Research Institute, Brno, Czech Republic 7 Public Health England, London, England 8 United States Department of Agriculture, Animal and Plant Health Inspection Services, Riverdale, MD, USA Correspondence Lia Koski, Division of Foodborne, Waterborne and Environmental Disease, Centers for Disease Control and Prevention, Atlanta, GA, USA. Email: [email protected] Abstract Reptile contact can result in zoonotic non‐typhoidal salmonellosis. In April 2018, Oregon Public Health Division contacted CDC about a cluster of four Salmonella se‐ rovar Fluntern (SF) illnesses in four states (OR, CA, IA, NY); patients reported con‐ tact with geckos, a popular reptile pet. PulseNet, the national molecular subtyping network of food‐borne disease surveillance, subsequently identified additional SF clinical isolates. Twelve cases in 11 states were identified; median age was 5 years (range: <1–58 years). Three patients were hospitalized; no deaths were reported. Of those with exposure information (n = 10), all reported reptile exposure; 9 (90%) speci‐ fied contact with leopard geckos. No common source of geckos was identified from reported purchase locations. Los Angeles County (LAC) health officials isolated SF from one patient's leopard gecko. Five reptile/gecko isolates were identified from the USDA National Veterinary Services Laboratories (NVSL) from 2015 to 2018. Five countries responded to an Epidemic Intelligence Information System post by PulseNet; reptile isolate sequence data were received from Czech Republic. A clini‐ cal case from England was identified through the National Center for Biotechnology Information pathogen detection pipeline; the patient did not report contact with leopard geckos. Whole genome sequencing analysis revealed substantial genetic di‐ versity between clinical and animal isolates; however, gecko and clinical isolates from LAC were highly related (1 allele difference). This investigation linking SF illnesses to leopard geckos highlights an important public health risk from pets. A better under‐ standing of how geckos are distributed by the pet industry in the United States could improve traceability to points of origin and mitigate Salmonella transmission at gecko breeders. Earlier NVSL reports of SF isolates from geckos suggest the risk of human SF infection from geckos is not new. This investigation demonstrates a need to edu‐ cate gecko breeders, retailers and gecko owners about the continued Salmonella in‐ fection risk from pet geckos. KEYWORDS outbreak investigation, reptiles, zoonotic salmonellosis Zoonoses Public Health. 2019;00:1–4. wileyonlinelibrary.com/journal/zph © 2019 Blackwell Verlag GmbH. | 1 2 | KOSKI et al. 1 | I NTRO D U C TI O N The Centers for Disease Control and Prevention (CDC) estimates 1.2 million non‐typhoidal Salmonella (NTS) illnesses occur in the United States every year (Hale et al., 2012). Zoonotic salmonellosis can occur when Salmonella is transmitted from an animal to a human, either through direct or indirect contact (The Center for Food Security & Public Health [CFSPH], 2013). It is estimated that 11% of all NTS infections in the United States result from zoonotic contact (Hale et al., 2012). Reptiles and amphibians are known asymptomatic carriers of NTS (CFSPH, 2013) and are an important source of NTS infections in the United States (Hale et al., 2012). Of all Salmonella serotypes, approximately 40% have been cultured predominantly from reptiles (Mermin et al., 2004). According to market research Impacts • Reptiles and amphibians continue to be an important source of zoonotic salmonellosis in the United States and worldwide. • This investigation linked Salmonella infections reported in the United States to leopard geckos and identified re‐ ports of salmonellosis linked to leopard geckos in certain European countries. • Whole genome sequencing results in this investigation suggest the need for research into host adaptation of Salmonella Fluntern in leopard geckos, given the broad genetic diversity among the clinical and animal isolates. statistics by the American Veterinary Medical Association (AVMA), 726,000 households in the United States owned pet lizards in 2012 (AVMA, 2012). Leopard Geckos, specifically, are popular pets be‐ Data from PulseNet were examined from 1 July 2017 through 1 cause they require minimal care, are easy to breed, and are poten‐ March 2018 to identify all Salmonella isolates uploaded as serotype tially long‐lived (De Vosjoli, Mazorlig, Klingenberg, Tremper, & Viets, Fluntern. A case was defined as isolation of Salmonella Fluntern from 2017). a patient during 1 July 2017 through 1 March 2018. State and local In April 2018, the Oregon Public Health Division contacted CDC health officials interviewed patients using a reptile‐ and gecko‐spe‐ about a cluster of four cases of Salmonella Fluntern in four states cific exposure questionnaire, which included questions about the (OR, CA, IA and NY), where ill people reported contact with geckos. species of reptile, type of gecko, exposure details and purchase in‐ Prior to these 2018 reports, only 90 other human and non‐human formation. Purchase locations reported by patients were contacted isolates of Salmonella Fluntern had been reported in the United for traceback information. State and local public health officials sam‐ States since 2002; 82 were isolated from human, clinical specimens pled geckos and reptile terraria from patients’ residences. and eight were animal isolates, of which six were specified to be PulseNet shared serotype information with PulseNet from geckos and two were noted to be ‘reptile, likely gecko.’ We in‐ International, and a notification was posted to the Epidemic vestigated to determine the source of the illnesses and to identify Intelligence Information System (EPIS) to query information from prevention measures. Although Salmonella Fluntern has been iden‐ public health agencies in over 50 countries. For strains that had been tified in reptiles and reptile terraria in certain European countries sequenced, raw sequence data files were shared with PulseNet via and in the United States and Canada (Ebani et al., 2005; Sockett a secure FTP site. Additionally, animal data regarding Salmonella & Rodgers, 2001; Tomastikova, Barazorda Romero, Knotek, & Fluntern were obtained from the United States Department of Karpiskova, 2017; Whitten, Bender, Smith, Leano, & Scheftel, 2015; Agriculture National Veterinary Services Laboratories (NVSL) and Wikström, Fernström, Melin, & Boqvist, 2014; Woodward, Khakhria, reviewed. WGS analysis using core genome multilocus sequencing & Johnson, 1997), this is the first investigation of Salmonella Fluntern typing (cgMLST) (Alikhan, Zhou, Sergeant, & Achtman, 2018) was illnesses linked to leopard geckos in the United States. performed to determine relatedness between clinical and animal isolates uploaded to the PulseNet National Database, NVSL, and 2 | M E TH O DS shared through PulseNet International. To assess strain relatedness, we constructed a cgMLST phylogenetic tree using BioNumerics v.7.6 (Applied Maths, http://www.appli​ed-maths.com). High‐quality sin‐ Salmonellosis is a notifiable condition in the United States (CSTE, gle nucleotide polymorphism (hqSNP) was used to validate the cgM‐ 2012). PulseNet is the national laboratory network for molecular LST results. These methods were used to assess strain relatedness, subtyping of Salmonella and other enteric pathogens, and PulseNet not to exclude or include cases in the investigation. International performs a similar role for enteric illnesses at the global level (Centers for Disease Control & Prevention PulseNet, 2016). Illness outbreaks are detected by monitoring geographic 3 | R E S U LT S and temporal clustering of Salmonella isolates with similar mo‐ lecular characteristics (Centers for Disease Control & Prevention Salmonella Fluntern is a rare serotype. Prior to 2018, Salmonella PulseNet, 2016). Molecular subtyping methods include serotyp‐ Fluntern isolates comprise 0.012% of all Salmonella isolates in the ing, pulsed‐field gel electrophoresis (PFGE) and whole genome PulseNet national database. Twelve cases in 11 states (CA, DE, FL, sequencing (WGS) (Centers for Disease Control & Prevention IA, MO, MT, NY (2), OK, OR, WI, WY) were identified during this PulseNet, 2016). investigation. Eight (67%) patients were female; median patient age | KOSKI et al. Allele differences 0–73 alleles; median = 47 Accession no. Source country FV1946 Czech Republic PNUSAS018756 USA PNUSAS033180 Source state Source type 3 Isolation date Animal unknown MT Human 2017-07-01 USA NY Human 2018-01-09 PNUSAS038744 USA FL Human 2018-02-27 S15-02044 USA GA Animal unknown  1–7alleles PNUSAS038745 USA CA Human 2018-02-07  1–7alleles PNUSAS040220 USA CA Animal 2018-04-23  1–7 alleles PNUSAS034985 USA IA Human 2018-02-08  1–2 alleles PNUSAS036175 USA OR Human 2018-02-20  1–2 alleles PNUSAS027845 USA WY Human 2017-10-18  1–2 alleles 440045 United Kingdom Human 2015-04-01 PNUSAS028872 USA NY Human 2017-10-26  indistinguishable S17-11420 USA CA Animal unknown  indistinguishable S18-02363 USA CA Animal unknown PNUSAS029308 USA Human 2017-08-29  indistinguishable FV1820 Czech Republic Animal unknown  indistinguishable FV1909 Czech Republic Animal unknown  7 alleles S15-02259 USA CO Animal unknown  7 alleles PNUSAS039230 USA MO Human 2017-11-29 S16-07392 USA CA Animal unknown PNUSAS034814 USA DE Human 2017-10-26 F I G U R E 1 cgMLST analysis for human and animal Salmonella Fluntern isolates identified in the investigation, constructed using BioNumerics v.7.6 (Applied Maths, http://www.appli​ed-maths.com) was 5 years (range: <1–58 years). Three patients were hospitalized, gecko isolates from 2017 to 2018 were indistinguishable from each and no deaths were reported. Of those patients with exposure in‐ other. Two Czech Republic reptile isolates were indistinguishable formation, all (n = 10) reported reptile exposure, and 9 of 10 (90%) from each other, but not closely related to any isolates from the specified contact with leopard geckos. Ill patients reported sev‐ United States (differed by 22–73 alleles). The third Czech Republic eral different purchase locations; however, no common source of isolate was unrelated to all other isolates in the analysis (differed by geckos was identified in this investigation. The Los Angeles County 18–73 alleles). Finally, a 2018 NVSL reptile isolate differed by seven Department of Public Health (LACDPH) isolated Salmonella Fluntern alleles from a 2017 clinical isolate from MO. These results were con‐ from one patient's pet leopard gecko. cordant with those from the hqSNP analysis. Five countries responded to the EPIS posting from PulseNet International: Czech Republic, Austria, Germany, Luxembourg and Slovakia. Four of these reports included human illnesses 4 | D I S C U S S I O N/CO N C LU S I O N linked to leopard gecko exposure, and WGS sequences of three reptile isolates from 2015 were received from the Veterinary This investigation of Salmonella Fluntern illnesses, linked to leopard Research Institute, Czech Republic. NVSL identified five lizard/ geckos, highlights an important public health risk of the transmission gecko isolates serotyped as Salmonella Fluntern with varying of Salmonella from a popular pet. No common source of geckos was PFGE patterns. Two were uploaded during the timeframe of this identified in this investigation. This suggests a potential need to bet‐ investigation, and three isolates were identified before this inves‐ ter understand how geckos are distributed by the pet industry in the tigation (2015–2017). Additionally, a 2015 clinical sequence from United States and internationally, as this could improve traceability the United Kingdom was identified using the National Center for of geckos to points of origin and mitigate Salmonella transmission at Biotechnology Information (NCBI) pathogen detection pipeline the breeder source. (NCBI, 2019); the patient did not report contact to leopard geckos, but the household did have pet fish. Whole genome sequencing analysis using cgMLST revealed a Certain Salmonella serotypes can be host‐adapted in different reptile species and become commensal organisms in the animal (Bäumler, Tsolis, Ficht, & Adams, 1998; Mermin et al., 2004). Host high degree of genetic diversity (0–73 alleles) among the 12 clinical adaptation of pathogenic strains of Salmonella poses a risk to pet and 9 animal isolates included in this investigation. While no true owners, as the animals appear healthy while carrying bacteria that clades were identified, some groups of isolates were more closely can result in human illness. Salmonella Fluntern is a rare serotype, related (<10 allele differences, Figure 1). These included the clinical and almost all animal isolates identified during and prior to this in‐ and gecko isolates from Los Angeles County, which differed by one vestigation in North America and Europe were from geckos. This allele from each other and 1–7 alleles from an Iowa clinical isolate. suggests that Salmonella Fluntern may be host‐adapted to geckos. Two clinical isolates from Wyoming and Oregon differed by 1–2 al‐ Bäumler et al., (1998) postulate that Salmonella adaptation to an leles from the clinical isolate from the United Kingdom. Two NVSL animal likely involves a large number of gene products, and more 4 | KOSKI et al. research is needed to understand which genetic changes account for adaptation. WGS analysis in this investigation revealed a high de‐ gree of genetic diversity among the clinical and animal isolates, with some more closely related groups among the strains. PulseNet con‐ siders a threshold of 10 allele differences for detecting WGS clus‐ ters by cgMLST, but recognizes that zoonotic outbreaks might have higher allele differences. Identification of Salmonella Fluntern gecko isolates as early as 2015 suggests the potential for transmission of Salmonella Fluntern from geckos to humans has been present for some time in the United States. Finally, this investigation indicates a need to better educate breeders, retailers and the public about the risk of Salmonella infection when owning and caring for pet leopard geckos. AC K N OW L E D G E M E N T S Khushbu Patel1. C O N FL I C T O F I N T E R E S T The authors have no conflict of interest to declare. DISCL AIMER The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention. ORCID Lia Koski https://orcid.org/0000-0002-7445-1686 REFERENCES Alikhan, N.‐F., Zhou, Z., Sergeant, M. J., & Achtman, M. (2018). A genomic overview of the population structure of Salmonella. PLoS Genetics, 14(4), e1007261. https​://doi.org/10.1371/journ​al.pgen.1007261 American Veterinary Medical Association (2012). U.S. pet ownership statistics. American Veterinary Medical Association. Retrieved from https​: //www.avma.org/KB/Resou​r ces/Stati​s tics/​P ages/​M arketresea​rch-stati​stics-US-pet-owner​ship.aspx Bäumler, A., Tsolis, R., Ficht, T., & Adams, L. (1998). Evolution of host adaptation in Salmonella enterica. Infection and Immunity, 66(10), 4579–4587. Center for Food Security and Public Health, Iowa State University (2013). Salmonellosis. 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Zoonoses Public Health. 2019;00:1–4. https​://doi.org/10.1111/zph.12647​