LETTERS health concern in industrialized and Author affi liations: Institute of Tropical 8. Ewers C, Grobbel M, Stamm I, Kopp PA, resource-poor settings. Few reports Medicine, Antwerp, Belgium (H. De Boeck, Diehl I, Semmler T, et al. Emergence of human pandemic O25:H4-ST131 CTX- J. Jacobs); Institut National de Recherche are available from Africa, although M-15 extended-spectrum-β-lactamase– hospital-associated ESBL producers Biomédicale, Kinshasa, Democratic Republic producing Escherichia coli among have been described in Cameroon of the Congo (B. Miwanda, O. Lunguya- companion animals. J Antimicrob Che- and the Central African Republic Metila, J.J. Muyembe-Tamfum); Maastricht mother. 2010;65:651–60. http://dx.doi. org/10.1093/jac/dkq004 University Medical Center, Maastricht, the (6,7). ESBL-producing bacteria have 9. Warren RE, Ensor VM, O’Neill P, Butler been recovered from different sources Netherlands (E. Stobberingh); and Cliniques V, Taylor J, Nye K, et al. Imported chicken in the community, including food Universitaires Université Catholique de meat as a potential source of quinolone- and companion animals (8,9), and 1 Louvain de Mont-Godinne, Yvoir, Belgium resistant Escherichia coli producing ex- tended-spectrum β-lactamases in the UK. (Y. Glupczynski) recent study from India reported that J Antimicrob Chemother. 2008;61:504–8. a substantial number of tap water DOI: http://dx.doi.org/10.3201/eid1806.111214 http://dx.doi.org/10.1093/jac/dkm517 samples were contaminated with 10. Walsh TR, Weeks J, Livermore DM, carbapenemase bla producing Toleman MA. Dissemination of NDM-1 NDM-1 References positive bacteria in the New Delhi envi- organisms (10). ronment and its implications for human Kinshasa is the second-largest 1. Okeke IN, Laxminarayan R, Bhutta ZA, health: an environmental point prevalence city in sub-Saharan Africa. In Duse AG, Jenkins P, O’Brien TF, et al. study. Lancet Infect Dis. 2011;11:355–62. Antimicrobial resistance in developing 2008, of its estimated 8.7 million http://dx.doi.org/10.1016/S1473-3099 countries. Part I: recent trends and current (11)70059-7 inhabitants, only 46%had access status. Lancet Infect Dis. 2005;5:481–93. http://dx.doi.org/10.1016/S1473-3099 to safe drinking water, and 23% Address for correspondence: Hilde De Boeck, had access to improved sanitation (05)70189-4 2. Vlieghe E, Phoba MF, Tamfun JJ, Ja- Institute of Tropical Medicine, Nationalestraat facilities according to the World cobs J. Antibiotic resistance among 155, Antwerp, Belgium; email: hdeboeck@itg. Bank. Opportunistic pathogens in bacterial pathogens in central Africa: a be drinking water and poor sanitary review of the published literature be- conditions may increase the risk of tween 1955 and 2008. Int J Antimicrob Agents. 2009;34:295–303. http://dx.doi. developing infectious enterocolitis org/10.1016/j.ijantimicag.2009.04.015 for consumers, especially for those 3. Clinical and Laboratory Standards Institute. who are immunocompromised. Performance standards for antimicrobial It can eventually lead to chronic susceptibility testing: 21st informational supplement. CLSI document M100-S21. intestinal carriage of multidrug- Wayne (PA): The Institute; 2011. resistant organisms. The presence 4. Endimiani A, Hujer AM, Hujer KM, Novel of ESBL producers in the intestinal Gatta JA, Schriver AC, Jacobs MR, et Chlamydiaceae fl ora could also lead to horizontal al. Evaluation of a commercial microar- ray system for detection of SHV-, TEM-, Disease in Captive transfer of drug resistance genes from CTX-M-, and KPC-type -lactamase genes commensal fl ora to enteric pathogens. in gram-negative isolates. J Clin Micro- Salamanders This emergence of ESBL-producing biol. 2010;48:2618–22. http://dx.doi. bacteria and further community- org/10.1128/JCM.00568-10 To the Editor: Although 2 major 5. Bradford PA. Extended-spectrum associated infections poses a public diseases of amphibians, chytridiomy- β-lactamases in the 21st century: char- cosis and ranavirosis, have been rela- threat, especially in low-resource acterization, epidemiology, and detec- countries where surveillance is tion of this important resistance threat. tively well studied, enigmatic amphib- suboptimal and empiric treatment Clin Microbiol Rev. 2001;14:933–51. ian disease and death not attributable http://dx.doi.org/10.1128/CMR.14.4.933- of invasive infections often includes to any of the known amphibian diseas- 951.2001 es frequently occur (1). We describe third-generation cephalosporins. 6. Gangoué-Piéboji J, Bedenic B, Koul- la-Shiro S, Randegger C, Adiogo D, an apparently new disease in salaman- Hilde De Boeck, Ngassam P, et al. Extended-spectrum- ders that is associated with a novel ge- Berthe Miwanda, β-lactamase-producing Enterobacteria- nus within the family Chlamydiaceae. ceae in Yaounde, Cameroon. J Clin Mi- The salamanders seen in our clinic Octavie Lunguya-Metila, crobiol. 2005;43:3273–7. http://dx.doi. Jean-Jacques Muyembe-Tamfum, org/10.1128/JCM.43.7.3273-3277.2005 belonged to 1 of the following species: Ellen Stobberingh, 7. Frank T, Arlet G, Gautier V, Talar- Salamandra corsica, the Corsican fi re Youri Glupczynski, min A, Bercion R. Extended-spectrum salamander (5 animals from 1 collec- β-lactamase-producing Enterobacteria- tion); Neurergus crocatus, the yellow and Jan Jacobs ceae, Central African Republic. Emerg Infect Dis. 2006;12:863. http://dx.doi. spotted newt (11 animals from 3 col- org/10.3201/eid1205.050951 lections); or N. strauchii, Strauch’s

1020 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 6, June 2012 LETTERS spotted newt (6 animals from 2 collec- in all 5 Corsican fi re salamanders; in mander species specimens (GenBank tions). All salamanders were captive 4/7, 1/3, and 1/1 yellow spotted newts; accession no. JN392919). These se- bred; housed in breeding colonies in and in 4/5 and 1/1 Strauch’s spotted quence differences point to the exis- private collections in Elsloo and Eind- newts. For taxon identifi cation, the tence of multiple strains with possible hoven, the Netherlands, Munich, Ger- 16S rRNA gene of the Chlamydiales host adaptation. many, and Brugge, Belgium; and 1–3 bacteria was amplifi ed and sequenced We determined the phylogenetic years of age. from the livers from 2 yellow spotted position of the novel taxon, named Disease was characterized by newts (1 from the collection in Elsloo, Candidatus Amphibiichlamydia sala- anorexia, lethargy, edema, and mark- the Netherlands and 1 from the collec- mandrae (online Technical Appendix), edly abnormal gait. Mortality rate tion in Munich, Germany), 1 Strauch’s identifi ed by using neighbor-joining was 100%. Animals in these collec- spotted newt, and 5 Corsican fi re sala- analysis with Kodon software (Ap- tions had no histories of disease. All manders. plied Maths, Sint-Martens-Latem, animals were in good nutritional con- The sequences shared >90% nt Belgium). The novel Chlamydiales dition. Necropsy did not yield any identity with the 16S rRNA gene of C. forms a distinct branch in the well- macroscopic lesions. All animals had abortus B577 (GenBank accession no. supported monophyletic clade with mild intestinal nematode or protozoan D85709) and therefore can be identi- the genera Chlamydia and Candidatus infections. Results of real-time PCRs fi ed as a member of the family Chla- Clavochlamydia salmonicola (family for iridoviruses in liver and skin (2) or mydiaceae (5). The closest 16S rRNA Chlamydiaceae) (Figure). Maximum Batrachochytrium dendrobatidis fun- similarity (92%) was observed with C. parsimony and unweighted pair group gus of skin (3) were negative for all psittaci strain CPX0308 (AB285329). with arithmetic mean analyses yielded animals. The sequence obtained from all spot- cladograms with the same topology We placed liver suspensions from ted newt species specimens was (results not shown). Previous reports of the dead salamanders on Columbia identical (GenBank accession no. members of the family Chlamydiaceae agar with 5% sheep blood and tryptic JN392920) but differed slightly (1%) in amphibians concerned species oc- soy agar and then incubated the sam- from that obtained from the fi re sala- curring in other vertebrate taxa as well: ples up to 14 days at 20°C. No con- sistent bacterial growth was observed. Histologic examination of 2 Corsican fi re salamanders and 1 yellow spot- ted newt revealed hepatitis in 1 of the Corsican fi re salamanders and the yel- low spotted newt. Hepatitis was char- acterized by high numbers of melano- macrophages and a marked infi ltration of granulocytic leukocytes. Immuno- histochemical staining for chlamydia (IMAGEN Chlamydia; Oxoid, Bas- ingstone, UK) showed cell-associated fl uorescently stained aggregates in liver tissue, suggestive of Chlamydi- ales bacteria. Transmission electron microscopic examination of the liver of a yellow spotted newt revealed intracellular inclusions containing particles matching the morphology of reticulate or elementary bodies of Chlamydiaceae (online Technical Ap- pendix, wwwnc.cdc.gov/EID/pdfs/11- 1137-Techapp.pdf). Figure. Topology of the novel amphibian Chlamydiaceae (Candidatus Amphibiichlamydia A PCR (4) to detect the 16S rRNA salamandrae) within the phylogenetic tree obtained by neighbor-joining and based on of all Chlamydiales bacteria, per- 16S rRNA gene data from representative species. Numbers show the percentage of times each branch was found in 1,000 bootstrap replicates. The tree has been rooted with formed on liver tissue samples from Verrucomicrobium spinosum as outgroup. Scale bar indicates nucleotide substitutions per all animals, yielded positive results site.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 6, June 2012 1021 LETTERS

C. psittaci, C. pneumoniae, C. abortus, 2. Mao J, Hedrick RP, Chichar VB. Molecular Novel Variant and C. suis (6–10). To our knowledge, characterization, sequence analysis, and taxonomic position of newly isolated fi sh of Beilong this member of the family Chlamydi- iridoviruses. Virology. 1997;229:212–20. aceae has been seen in amphibians, but http://dx.doi.org/10.1006/viro.1996.8435 Paramyxovirus not in other vertebrate hosts. The 16S 3. Boyle DG, Boyle DB, Olsen V, Morgan in Rats, China rRNA analysis showed this taxon to be- JAT, Hyatt AD. Rapid quantitative detec- tion of Chytridiomycosis (Batrachochy- long to a clade with Candidatus Clavo- trium dendrobatidis) in amphibian sam- To the Editor: In 2003, two chlamydia salmonicola, a taxon found ples using real-time Taqman PCR assay. cDNA strands were identifi ed in a in fi sh. The phylogenetic position of the Dis Aquat Organ. 2004;60:141–8. http:// human mesangial cell line during novel taxon in the family Chlamydia- dx.doi.org/10.3354/dao060141 experimental screening for genes 4. Everett KDE. Chlamydia and Chlamydi- ceae thus roughly refl ects the phyloge- ales: more than meets the eye. Vet Mi- upregulated by angiotensin II (1). netic relation between the host species, crobiol. 2000;75:109–26. http://dx.doi. Sequence analysis showed that the providing evidence for host–bacterium org/10.1016/S0378-1135(00)00213-3 strands were homologous to the matrix, co-evolution in the family Chlamydi- 5. Everett KDE, Bush RM, Andersen AA. fusion, and phosphoprotein genes Emended description of the order Chla- aceae. mydiales, proposal of Parachlamydi- of paramyxoviruses, suggesting the Although the results obtained aceae fam. nov. and Simkaniaceae fam. possibility of a novel paramyxovirus are not conclusive with regard to the nov., each containing one monotypic (2,3). Subsequent research found that pathogenic potential of this novel ge- genus, revised taxonomy of the family these sequences, believed to originate Chlamydiaceae, including a new genus nus and species of Chlamydiales, we and fi ve new species, and standards for from human kidney mesangial cell were not able to attribute the clinical the identifi cation of organisms. Int J Syst lines, were not amplifi able from such signs to any known disease. We there- Bacteriol. 1999;49:415–40. http://dx.doi. cell lines or human kidney samples fore suggest that we discovered a nov- org/10.1099/00207713-49-2-415 but were amplifi able from a rat kidney 6. Berger L, Volp K, Mathews S, Speare R, el bacterial taxon with possible con- Timms P. Chlamydia pneumoniae in a mesangial cell line (4). Isolation and siderable impact on amphibian health. free-ranging giant barred frog (Mixophyes complete genome sequencing of the iterates) from Australia. J Clin Microbiol. virus confi rmed that it was a novel 1999;37:2378–80. Acknowledgments paramyxovirus of the subfamily 7. Blumer C, Zimmermand DR, Weilenmann We are grateful to Jean Euzéby for R, Vaughan L, Pospischil A. Chlamydiae Paramyxovirinae, named Beilong support with the nomenclature. We thank in free-ranging and captive frogs in Swit- virus (BeV). Rob Keulers for his cooperation. zerland. Vet Pathol. 2007;44:144–50. BeV is most closely related to http://dx.doi.org/10.1354/vp.44-2-144 J virus, discovered in autoculture 8. Howerth EW. Pathology of naturally- of kidney tissue from a moribund An Martel, Connie Adriaensen, occurring chlamydiosis in African clawed house mouse, and Tailam virus from Sergé Bogaerts, frogs (Xenopus laevis). Vet Pathol. 1984;21:28–32. Sikkim rats (5,6). Because J virus and Richard Ducatelle, 9. Newcomer CE, Anver MR, Simmons JL, Tailam virus were found to originate Herman Favoreel, Wilcke RW, Nace GW. Spontaneous and in rodents and BeV was amplifi able Sandra Crameri, Alex D. Hyatt, experimental infections of Xenopus laevis from a rat kidney mesangial cell Freddy Haesebrouck, with Chlamydia psittaci. Lab Anim Sci. 1982;32:680–6. line, we hypothesized that BeV was and Frank Pasmans 10. Reed KD, Ruth GR, Meyer JA, Shukla a novel paramyxovirus originating Author affi liations: Ghent University, Merel- SK. Chlamydia pneumonia infection in a in rats. To test this hypothesis, we beke, Belgium (A. Martel, C. Adriaensen, breeding colony of African clawed frogs (Xenopus tropicalis). Emerg Infect Dis. conducted a territorywide molecular R. Ducatelle, H. Favoreel, F. Haesebrouck, 2000;6:196–9. http://dx.doi.org/10.3201/ epidemiologic study of rats and other F. Pasmans); Waalre, the Netherlands (S. eid0602.000216 mammals to evaluate this novel Bogaerts); Commonwealth Scientifi c and paramyxovirus. Industrial Research Organisation, Geelong, Address for correspondence: An Martel, We tested 4,130 samples from Victoria, Australia (S. Crameri, A.D. Hyatt) Department of Pathology, Bacteriology and 1,398 animals collected from various Avian Diseases, Faculty of Veterinary Medicine, DOI: http://dx.doi.org/10.3201/eid1806.111137 locations in Hong Kong, People’s Ghent University, Salisburylaan 133, B-9820 Republic of China, during September Belgium; email: [email protected] References 2008–August 2009 (Table). These included 480 kidney, spleen, 1. Daszak P, Berger L, Cunningham AA, All material published in Emerging Hyat AD, Green DE, Speare R. Emerg- Infectious Diseases is in the public respiratory swab, and anal swab ing infectious diseases and amphibian domain and may be used and samples from 120 asymptomatic rats population declines. Emerg Infect Dis. reprinted without special permission; (105 brown rats [Rattus norvegicus] 1999;5:735–48. http://dx.doi.org/10.3201/ proper citation, however, is required. eid0506.990601 and 15 black rats [R. rattus]). To

1022 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 6, June 2012 Article DOI: http://dx.doi.org/10.3201/eid1806.111137 Novel Chlamydiaceae Disease in Captive Salamanders

Technical Appendix

Description of Candidatus Amphibiichlamydia salamandrae

Candidatus Amphibiichlamydia salamandrae [Amphibii chlamydia N.L. n. Amphibia name of host class; L. fem. n. Chlamydia name of bacterial taxon; N.L. fem. n. Amphibiichlamydia Chlamydia from an amphibian; salamandra’e. L. gen. n. salamandrae of a salamander]

The provisional taxon “Candidatus Amphibiichlamydia salamandrae” contains intracellular bacteria that infect salamanders of the genera Neurergus and Salamandra in freshwater or terrestrial environments. The 16S rRNA gene of Candidatus Amphibiichlamydia salamandrae has been deposited in the GenBank under accession nos. JN392920 and JN392919. The 16S rRNA gene shows phylogenetic affinity toward the family Chlamydiaceae.

Technical Appendix Figure. Transmission electron micrograph of a liver section of a yellow spotted newt (Neurergus crocatus) showing an intracellular vacuole containing elementary bodies and reticulate bodies of Chlamydia-like organisms. Original magnification ×4,000.

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