ORAL MICROBIOTA OF PATAGONIAN OF (): FABLE TO FACTS

NORA R. IBARGÜENGOYTÍA 1, PHILIP S. BIRD 2, FRANCISCO A. UZAL 3 & ANA L. CIPOLLA 2

n o r a i @ b a r i l o c h e . c o m . a r

Cuad.R E S herpetol.,U M E N 18 (2): 37 41, 2004 (2005) Diplolaemus

Cuad. herpetol., 18 (2): 37–41,– 2004 (2005)

1 Diplolaemus D. darwini Diplolae- mus 2

3 37 Clostridium perfigrens 4 Centro Regional Universitario Bariloche. Universidad Nacional del Comahue. Unidad Postal Universidad del Comahue, Bariloche,Staphylococus 8400, Río warneriNegro, Argentina.Clostridium Consejo bifermentans, Nacional de ClostridiumInvestigaciones perfringens científicas y técnicas.Stoma- tococcus(CONICET). muscilaginosus. Phone: 0054-2944-423374/428505. Fax: 0054-2944-422111. Oral Biology and Pathology, School of Dentistry, The University of Queensland, St Lucia, Queensland 4072, Australia. Diplolaemus California Health and Food Safety Laboratory. Faculty of Veterinary Medicine, University of Cal- ifornia, Davis. 105 WDiplolaemus Central Ave., San Bernardino CA 92408. USA. . Instituto Nacional de Tecnología Agropecuaria- Estación Experimental Balcarce-Area de Producción Animal- Laboratorio de Bacteriología. Ruta Nac. 226 - Km 73,5, Balcarce (7620), Pcia Bs. As.

A B S T R A C T Diplolaemus

Diplolaemus . — Existe la creencia popular de que los lagartos del género (Leio- D. darwini Diplolaemus sauridae) de Patagonia, Argentina, son capaces de conferir mordeduras venenosas que pueden causar inflamación e incluso la muerte de animales de granja o salvajes. No obstante, no existe evidencia de la existencia de glándulas de veneno en estos lagartos. La inflamación y absceso pro- vocado como resultado de las heridas de mordedura podría ser causado por agentes infecciosos. Clostridium perfringens Este es el primer estudio acerca de la microbiota oral de los lagartos del género de Staphylococcus warneri, Clostridium perfringens, Clostridium Argentina. Para este trabajo se analizó un espécimen de y tres individuos de bifermentans Stomatococcus muscilaginosus «forma alto patagonica». Las muestras de hisopados de boca de los lagartos fueron incubados aeróbica y anaeróbicamente, algunos de ellos fueron subcultivados en agar de sangre ovina al 5% Diplolaemus e incubados nuevamente. Todas las colonias representativas observadas fueron subcultivadas para su purificación y sujetas a tests bioquímicos. La bacteria fue determinada Diplolaemus por medio de la técnica de reacción en cadena de la Polimerasa. Se identificaron cuatro especies de bacterias: , y Estas especies son conocidas como agentes etiológicos de numerosas in- fecciones de humanos y animales. Si bien estas bacterias representan sólo un pequeño número de las posibles bacterias aisladas de la cavidad oral de , están indicando la presencia de patógenos humanos y animales. o Palabras clave: , lagartijas, microbiología, enfermedades

. — Lizards of genus (Leiosauridae) from Patagonia, Argentina are believed to have «poisonous» bites that can cause inflammation and death of farm and wild . However there is no evidence of poisonous glands in these lizards. It is proposed that the inflammation and abscesses resulting from these bite wounds are caused by infectious agent(s). This is the first study of the oral microbiota of lizards from Argentina. One specimen of and three individuals of «forma alto patagonica» were used for the present work. Swabs were taken from the mouths of the lizards, subcultured onto 5% sheep blood agar plates, incubated at 37 C for 48 hours aerobically and anaerobically. All representative colonies observed were subcultured for purity and subjected to conventional biochemical tests. Isolates identified as were typed by a polymerase chain reaction technique. Four of bacteria were identified as and . These species are known etiologic agents of a number of human and animal infections. While these bacteria represent only a small number of possible isolates from the oral cavity of species nevertheless it indicates that human and animal pathogens are present in their oral cavity. Key words: , lizards, microbiology, diseases. et al. Diplolaemus

Diplolaemus

Diplolaemus Diplolae- mus darwini, Diplolaemus bibroni Diplolaemus leopardinus

D. darwini D. bibroni Diplolaemus

Diplolaemus BARGÜENGOYTÍA et al

Diplolaemus N. R. I : Oral Microbiota of Lizards D. darwini Diplolaemus

38 The lizards (matuastos) caused by infectious agent(s) present in are distributed throughout Patagonia, their mouths and transmitted to their Argentina and Chile´s southernmost re- prey by the bite of these lizards we per- gion. The genus (Leiosau- formed the present study. ridae) consists of three species ( Also, the oral microbial flora can be and involved in serious infections of several ) and two forms, captive lizards such as agamids, chame- not yet recognized as species, which are leons, and iguanas, producing considered as hybrids of and periodontalº disease which is an impor- . These two forms are call tant cause of disease and death of these «forma alto-patagónica» and animals (McCracken and Birch, 1994; «forma sudmendocina», re- McCracken ., 1999). However, infor- spectively (Cei, 1986, 1993). They are mation about the oral microbial flora of very powerful and aggressive lizards lizards in their nature environment is with a large head and strong wide jaws. scanty (Ciofi, 1999).This preliminary They are up to 12 cm snout-vent length study was therefore designed to deter- (Cei, 1986). mine if the lizards of the genus Clostridium per- At present, there is a general con- carry pathogenic bacteria fringens sensus that the way local people per- in their oral cavity. ceive and use their natural environment One specimen of and plays an important role in the conserva- three individuals of «forma tion of resources and its consequent alto patagonica» were captured in the et al. sustainable development. The Conven- field and transported alive to the labo- tion of Biological Diversity recognizes ratory. The lizards were clinically exam- that biodiversity will not be conserved ined (including the mouth) and a sam- without understanding how humans in- ple was taken from the mouth of each teract with the natural environment using a swab moistened with (Ladio and Lozada, 2000). Individuals sterile saline. The swabs were inoculat- have the strongest affinities with family ed onto 5% sheep blood agar plates, members and select the cultural vari- and incubated aerobically and anaerobi- ants provided by them thus generating cally. Swabs were also inoculated into a vertical transmission from parents to cooked meat broth (CMB), incubated children. Under these circumstances, anaerobically and then subcultured onto most ecological constraints are intimate- 5% sheep blood agar plates and incubat- ly associated with social factors, both ed again. All incubations were carried contributing to an adaptive solution out at 37 C for 72 h and all representa- (Ladio and Lozada, 2000). There are his- tive colonies observed on the agar torical records that show there was a plates were subcultured for purity and belief at the end of XIX century among subjected to conventional biochemical local people in Patagonia that «matuas- tests as previously described, including tos» were poisonous (Musters, 1997) and carbohydrate fermentation, heamolysis this belief still continues to the present of sheep erythrocytes and lecithinase day. Credence for this is that the liz- activity (Sterne and Batty, 1978). Iso- ards´ bite produce abscesses with result- lates were identified as ing massive inflammation, sometimes by a polymerase chain reaction resulting in the death of dogs and (PCR) technique to amplify the alpha, sheep (Musters, 1997). However, no poi- beta, epsilon and iota toxin genes of sonous glands have been found in any of this microorganism, as previously re- the «matuastos» (Cei, 1986; 1993). ported (Uzal , 1997). Therefore to test the hypothesis that All the lizards used in this study were the abscesses and inflammation are clinically normal when swabs were ob- Staphylococus warneri D. darwini

Clostridium bifer- mentans, C. perfringens Stomatococ- cus muscilaginosus Diplolaemus C. perfringens Varanus komodoensis

Diplolaemus

S. warneri

et V. komodoensis Cuad.al herpetol., 18 (2): 37 41, 2004 (2005) et al – et al

Diplolaemus

Diplolaemus39 tained from their mouths. No gross ab- ism is ubiquitous and it can be found in et al S. mucilaginosus normalities were observed in the mouth soil and feces of clinically normal ani- of any of the animals investigated. mals and human beings, is considered was isolated an opportunistic pathogen that can pro- Diplolaemus from . No other aerobic or liferate and produce disease under ap- anaerobic microorganisms were cultured propriate conditions. The detection of from this individual. the alpha toxin gene in the isolated et al et al C. bi- and strain is an indicator that this was a fermentans were isolated from potential pathogen that carries a viru- et al the three individuals of «for- lence factor in its genome. et al ma alto patagonica». The The oral cavity of the Komodo sauri- et isolated was typed as type A by PCR. an lizard, , sup- al Bacteria isolated from the oral cavity ports a large numbers of bacteria and it et al of in this study are etiolog- is thought that bite wounds inflicted by ical agents of animal and human diseas- this lizard can result in serious infec- et al C. perfringens es. has been associated to a tion and septicemia that may eventually number of human and animal infec- kill the prey (Ciofi, 1999). In one study tions, including endocarditis (Fabbri of the oral cavity of , ., 1990), septicaemia, osteomyelitis approximately 50 different bacterial spe- (Bryan ., 1987) and urinary tract cies wereACKNOWLEDGEMENTS identified and at least seven infections (Leighton and Little, 1986). of these were highly pathogenic (Arnett, This microorganism is also invasive in 1998). Our results, albeit from a re- cancer patients (Eng ., 1982) and duced number of animals and isolates, elicits the release of a factor that show that pathogenic bacteria are found strongly activates the human immunode- in the mouth of the liz- ficiency virus type 1 (HIV1) influencing ards, as well. Nevertheless, the number the progression of HIV1 infection (Kle- of bacteria species found in banoff ., 1994). is lizards were much smaller than that a normal inhabitant of the human oral found in the Komodo dragon. Differenc- cavity, but it can also produce septic es in the diet (the Komodo dragon is shock, meningitis, acute respiratory dis- carnivorous while the liz- tress syndrome, endocarditis and cathe- ards are insectivorous) and habitat (Ko- ter-related infections in humans (Ascher modo Island is a tropical environment ., 1991; Gruson ., 1998). with a high rainfall rate, while most of produces necrotizing pneu- Patagonia is a cold semi-desert) are monia, empyema (Jonsson ., 1983), probably responsible for the difference in endocarditis (Kolander ., 1989), liv- oral microflora between these , er and abdominal abscess (Nachman among others. The present results indi- ., 1989; Rechtman and Nadler, 1991), cate that, at least potentially, the bites septic arthritis (Nolan ., 1972) and of the matuastos may result in trans- osteomyelitis in human beings (Scanlan mission of infectious agent(s) causing ., 1994). type A is serious infection. More research is cur- commonly found in the soil and intesti- rently under way to further test this nal tract of humans and other animals hypothesis. (Stratonova, 1969; Uzal and Marcellino, 2002); it produces gas gangrene and food poisoning in humans (Chakrabarty and Boro, 1981) and it is also responsi- ble for enterotoxaemia and malignant I wish to express my gratitude to my oedema in several animal species (Mc- mother, Adelina E. Pérez, and to Víctor Clane, 1996). Although this microorgan- Cussac for their helpful comments of et al. Diplolaemus

Clostridium perfringens Ze- ntralblatt fur Bakteriologie, Mikro- biologie und Hygiene

Scientif- ic American

BARGÜENGOYTÍA

Journal of Clinical Mi- crobiology

N. R. I : Oral Microbiota of Lizards LITERATURE CITED Pediatria Vara- Medica e Chirurgica nus komodoensis

IOFI

40 Stomato- the manuscript and to Alejandro Scolaro alence of food-poisoning (entero- Stomatococcus muci- coccus mucilaginosus to help with the systematic. I want also toxigenic) laginosus to express my special thanks to my NG type A inANG blood andERSON fish meal.IEHN Hematology daughter Manuela Delfina Martinez. RMSTRONG Southern Medical Journal and Cell Therapy This work was partially supported by 172: 427-433. Universidad Nacional del Comahue, Ar- C , C. 1999. The Komodo Dragon: On gentina and the Consejo Nacional de In- a few small islands in the Indone- Sta- Bacillus vestigaciones Científicas y Técnicas. Se- sian archipelago, the world’s larg- phylococcus warneri cereus Clostridium bifermen- ría en vez de: I wish to express my gra- ABRI est lizardACCHELLA reigns supreme.ONDINO Diagnostic tans American Review of Respira- titude to Víctor Cussac for his helpful ORONI 280:ONRAD 64-71. Microbiology and Infectious Dis- tory Disease comments of the manuscript and to Ale- E , R.; C. W ; A. P ; T. K eases jandro Scolaro to help with the syste- & D. A . 1982. Species matic. This work was partially suppor- identification of coagulase-negative tedRNETT by Universidad Nacional del Coma- staphylococcal isolates from blood hue, Argentina and the Consejo Nacio- RUSONcultures. ILBERT IGNEUX Museo Regionale nal de Investigaciones Científicas y Téc- ARGAS 15: UISSET439-442. EXIER di Scienze Naturali Torino nicas. F , A.;OIRON A. T EIFFERS; V. M BIKPI ; C.E Monogr., MNISSAN & E. CARDINAUD. 1990. Non- SCHER ASH BICK epidermidis coagulase-negative sta- HITE phylococci in infectious diseases of the compromised host. Museo Clostridi- A , J. 1998. Komodo Dragons, 12: 233-236. Regionale di Scienze Naturali um bifermentans , killers or life- G , D.; G. H ; A. P ; F. Torino Monogr. Archives of Internal savers? 22nd International Hepe- ONSSONV ; O. LARRIDGEG ; J. T OUNG; J. Medicine RYANtological Symopsium,ARISI Cincinnati,RIKE B ; J. R ; G. G -B - Ohio, USA. & J. C . 1998. Se- A , D.; M. B ; C. Z & C. vere infection caused by W . 1991. in a neutro- catheter-related infection penic patient: case report and re- in an adolescent with osteosarco- LEBANOFFview of the literature.AZAZI AN EI ma. 84: OORHIS CHLECHTE 40: 167-169. 409-410. J , S.; J. C & E. Y . B , C.; J. P & D. S . 1987. 1983. Necrotizing pneumonia and Vertebral osteomyelitis due to empyema caused by attributed to and a Hickman catheter. . EI 127: 357-359. 8: 57-59. KOLANDER , S.; F.OSGROVE K ; W. OLAVIV C , J. M. 1986. Reptiles del centro, V & K. S . 1994. Ac- centro-oeste y sur de la Argenti- tivation of the human immunodefi- na, Herpetofauna de las zonas ari- ciency virus long terminal repeat das y semiaridas. in THP-1 cells by a staphylococcal HAKRABARTY ORO extracellular product. Proceedings Italy pp. 4: 527 pp. of the National Academy of Sci- C , J. M.1993. Reptiles del noroeste, ences USA 91: 10615-10619. nordeste y este de la Argentina., K , S.; E. C & A. M . Herpetofauna de las selvas sub- 1989. Clostridial endocarditis. Re- tropicales, Puna y Pampas. port of a case caused by and review of , Italy. 14: 949 pp. the literature. C , A. & B. B . 1981. Prev- 149: 455-456. Hu- Clostridium bifermentans man Ecology The Journal of Bone and Joint Surgery American

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Clostridium bifermentans American Journal of Clinical Pathology Journal of Clinical Microbiology

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