Raoultella ornithinolytica Isolation in Cloacal Microbiota of Tinamus solitarius : Preliminary Data

Raoultella ornithinolytica is a gram-negative aerobic bacterium belonging to Enterobacteriaceae family, an emerging pathogen that causes several pathogenic conditions in man, with little veterinary importance; however, its identification is underestimated by conventional laboratory techniques. The present study reports the identification of R. ornithinolytica in Tinamus solitaries, during a routine sanitary evaluation of aerobic enterobacteria in cloacal microbiota of birds belonging to the Güira Oga Center, Puerto Iguazu, Argentina. The sample was preliminary classified as Klebsiella spp.; however, after the use of the MALDI-TOF MS technique it was identified as R. ornithinolytica. The sample was submitted to an antimicrobial susceptibility test, where it showed a similar pattern profile as reported in the literature, with resistance to ampicillin and other β-lactam antibiotics. It is possible that Raoultella spp are more common in birds as it is reported. Therefore, review studies on bacteria collections of avian origin, as well as cases with confirmation of Klebsiella, should be deeply evaluated in laboratorial routine, mainly due to the pathogenic potential of R. ornithinolytica for Poultry, as well as for public health.


IntRoduCtIon
Raoultella ornithinolytica is a gram-negative aerobic bacterium belonging to the Enterobacteriaceae family is commonly present in the aquatic environment (Park et al., 2011;Jong et al., 2013).The genus Raoultella are composed by species belonging to the genus Klebsiella, differentiated by molecular techniques and by having a distinct behavior such as growing at low temperatures, and by using sorbose as carbon source (Drancourt et al., 2001).
Due to the similarities between Raoultella and Klebsiella genus it is impossible to distinguish both by conventional biochemical techniques (de Jong et al., 2013), being identified only by advanced identification techniques.
The present study reports the identification of Raoultella ornithinolytica sample in a healthy Tinamus solitarius during a sanitary evaluation of aerobic enterobacteria in routine cloacal microbiota from a group of twenty one birds, belonging to the Güira Oga Center, Puerto Iguazu, Argentina.
Samples were collected through the swab technique.Prior to the collection cloacal asepsis was performed (with alcohol 70°) in order to avoid contamination.Material collection was performed individually by using sterile swabs.Swabs were inserted in the cloaca through soft circular movements in order to maximize the contact with the cloacal mucosa.After collection, each swab was conditioned in flasks containing Stuart media and kept refrigerated at 8 °C until processing.
The samples were processed in the Laboratory of Microbiology at Universidade do Oeste de Santa Catarina (UNOESC, Xanxerê, SC, Brazil) where the swabs were incubated in peptone water at 40 °C for 24 hours.One mL of each peptone broth samples was added to tubes containing Brain and Heart Broth (BHI) and Tetrathionate Broth (TB), incubated again at 40°C during 24 hours.Then, the samples were seeded on MacConkey and Xylose-Lysine-Deoxycholate agar, incubated at 40 °C for 24 hours.All the different colonies that grew in the different media were selected and, after isolation of each strain, frozen at -80 °C in nutrient broth plus 10% glycerol and sent to the Ornithology Laboratory of Faculdade de Medicina Veterinária e Zootecnia of Universidade Estadual Paulista (FMVZ-UNESP, Botucatu, SP, Brazil) for the biochemical pre-screening assay of the samples, composed of urea, malonate broth, Sulfito-Indol-Motility medium, triple sugar and iron agar, and methyl red and Voges-Proskauer tests.
The samples were then sent to the Laboratório de Medicina Aviária of Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo (FMVZ-USP, São Paulo, SP, Brazil), being submitted to the matrix assisted laser desorption technique -MALDI-TOF MS for confirmation of the isolated species.
The technique confirmed pre-identified samples in genus Escherichia, Enterobacter, Serratia, Proteus and Klebsiella; however, one of the pre-identified samples has proved to be Raoultella ornithinolytica.R. ornithinolytica sample was subjected to antibiogram sensitivity tests in order to assess the resistance profile, according to Bauer et al. (1966), using commercial antibiotic impregnated disks (Sensifar-Vet Cefar ® , Cefar Diagnóstica Ltda, São Paulo, SP, Brazil).The sample was challenged by 26 distinct antibiotics (Table 1), demonstrating resistance to only seven antibiotics (ampicillin, bacitracin, enrofloxacin, erythromycin, spiramycin, penicillin and rifampicin) intermediate sensitivity only to amoxicillin.
R. ornithinolytica is known as one of the bacteria in charge for scombroid poisoning, a toxic condition due to the ingestion of fish with high levels of histamine (Masashi et al., 2002); however, in the last decade it was considered as an emerging pathogen in humans (Seng et al., 2016) involved in urinary tract infection (García-Lozano et al., 2013;Nakasone et al., 2015), septicemia (Kaya et al., 2014;Sueifan et al., 2016), bacteremia (Mau & Ross, 2010;Hadano et al., 2012;Haruki et al., 2014;Sekowska et al., 2015), peritonitis (Sibanda, 2014), enteric fever (Morais et al., 2009), biliary infection (Cleveland et al., 2014;de Jong et al., 2014), infected "diabetic foot" (Solak et al., 2011;Kabbara & Zgheib, 2015) and artritis (Venus et al.,2016).However, cases of infection were always related to some factor of primary malignancy or immune deficiency (Chun et al., 2015;Boattini et al., 2016).The isolation of this pathogen in animals is uncommon and there are few records in birds.As in the present case, it has already been described in a cloacal microbiota of a healthy vulture (Sala et al., 2016) and in a case of hepatitis in Ring-Neck (Gonzales-Lama & Lupiola-Gomez, 2007).Nevertheless, both cases were reported in Europe.Thus, this is the first report of R. ornithinolytica isolation in a wild bird in South America.
Regarding the antimicrobial susceptibility profile, as observed by Morais et al. (2009), Mau & Ross (2010), García-Lozano et al. (2013), Kaya et al. (2015), Sekowska et al. (2015), Ponce-Alonso et al. (2016) and Seng et al. (2016), there was low sensitivity to β-lactam antibiotics.This occurs due to the presence of chromosomal resistance genes for the production of β-lactamases (Walckenaer et al., 2004).The macrolide resistance observed in this study was also expected since, according to Ito et al. (2005), this pharmacological group is mainly indicated for Grampositive bacteria and the efficiency is widely variable in Gram-negative bacteria.As mentioned in these studies, the resistance to other antibiotics are varied; thus, not showing a specific pattern.
In general, the incidence of Raultella ornithinolytica is underestimated due to the imprecision of conventional phenotypic identification methods, generating uncertainties as to its pathogenicity (Ponce-Alonso et al., 2016), mainly due to the similarities with species of the Klebsiella genus, such as K. pneumonie and K. oxytoca.The only way to achieve reliable identification of R. ornithinolytica and other species of Raoultella is applying supplemental biochemical tests and/or by using the MALDI-TOF MS technique.
It is possible that species of the genus Raoultella are common in birds.Cases of bacterial collection of avian origin, as well as the confirmation in cases involving samples Klebsiella in birds should be more investigated as potentially being Raoultella, in laboratorial routine, mainly due to its pathogenic potential for birds, as well as for public health.Therefore, it will be possible to determine the importance of birds in the epidemiological chain of this poorly studied bacterium.