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

GA Marietto-Gonçalves EL Lima BA Nagayoshi AA Tonin T Knöbl RL Adreatti FilhoAbout the authors

ABSTRACT

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.

Keywords:
Raoultella; Enterobacteria; microbiota; wild birds; 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., 2011Park JS, Hong KH, Lee HJ, Choi SH, Song SH, Song KH, et al. Evaluation of three phenotypic identification systems for clinical isolates of Raoultella ornithinolytica. Journal of Medical Microbiology 2011;60(4):492-499.; 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., 2001Drancourt M, Bollet C, Carta A, Rousselier P. Phylogenetic analyses of Klebsiella species delineate Klebsiella and Raoultella gen. nov., with description of Raoultella ornithinolytica comb. nov., Raoultella terrigena comb. nov. and Raoultella planticola comb. nov. International Journal of Systematic and Evolutionary Microbiology 2001;51:925-932.).

It is an emerging pathogenic bacterium that presents some virulence factors such as capsule, CFA/I and CFA/II colonization factors, as well as production of siderophores, histamine and bacteriocins (Podschun et al., 1993Podschun R, Sievers D, Fischer A, Ullmann U. Serotypes, hemma-gglutinins, siderophore synthesis, and serum resistance of Klebsiella isolates causing human urinary tract infections. Journal of Infectious Diseases 1993;168(6):1415-1421.; Podschun et al., 1998; Kanki et al., 2007; Al-Hulu et al., 2009Al-Hulu SM, Al-Charrakh AL, Al-Saadi MAK. Isolation and characterization of Raoultella ornithinolytica from clinical specimens in Hilla city, Iraq. Medical Journal of Babylon 2009;7(4):42-47.; Seng et al., 2016Seng P, Boushab BM, Ramain F, Gouriet F, Bruder N, Martin C, et al. Emerging role of Raoultella ornithinolytica in human infections: a series of cases and review of literature. International Journal of Infect Diseases 2016;45(2016):65-71.).

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. (1966Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 1966;45(4):493-496.), 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.

Table 1
Antimicrobial susceptibility profile of Raoultella ornithinolytica.

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., 2002Masashi K, Tomoko Y, Teizo T, Tadayoshi S. Klebsiella pneumoniae produces no histamine: Raoultella planticola and Raoultella ornithinolytica strains are histamine producers. Applied and Environmental Microbiology 2002;68(7):3462-3466.); however, in the last decade it was considered as an emerging pathogen in humans (Seng et al., 2016Seng P, Boushab BM, Ramain F, Gouriet F, Bruder N, Martin C, et al. Emerging role of Raoultella ornithinolytica in human infections: a series of cases and review of literature. International Journal of Infect Diseases 2016;45(2016):65-71.) involved in urinary tract infection (García-Lozano et al., 2013García-Lozano T, Plá FJP, Aznar-Oroval E. Raoultella ornithinolytica in urinary tract infections. Clinical and microbiological study of a series of 4 oncologic patients. Medicina Clinica 2013;141(3):138-139.; Nakasone et al., 2015Nakasone ES, Kaneshiro R, Min K, Tokeshi J. Emergence of Raoultella ornithinolytica on O'ahu: a case of community-acquire R. Ornithinolytica urinary tract infection. Hawai'i Journal of Medicine & Public Health 2015;74(5):174-175.), septicemia (Kaya et al., 2014; Sueifan et al., 2016Sueifan M, Moog V, Rau E, Eichenauer T. Sepsis caused by Raoultella ornithinolyticain an immunocompetent patient. Der Anaesthesist 2016;65(2):120-133.), bacteremia (Mau & Ross, 2010Mau N, Ross LA. Raoultella ornithinolytica bacteremia in an infant with visceral heterotaxy. Pediatric Infectious Disease Journal 2010;29(5):477-478.; Hadano et al., 2012Hadano Y, Tsukahara M, Ito K, Suzuki J, Kawamura I, Kurai H. Raoultella ornithinolytica bacteremia in cancer patients: report of three cases. Internal Medicine 2012;51(22):3193-3195.; Haruki et al., 2014Haruki Y, Hagiya H, Sakuma A, Murase T, Sugiyama T, Kondo S. Clinical characteristics of Raoultella ornithinolytica bacteremia: a case series and literature review. Journal of Infection and Chemotherapy 2014;20(9):589-591.; Sekowska et al., 2015Sekowska A, Dylewska K, Gospodarek E, Bogiel T. Catheter-related blood stream infection caused by Raoultella ornithinolytica. Folia Microbiologica 2015;60(6):493-495.), peritonitis (Sibanda, 2014Sibanda M. Primary peritonitis caused by Raoultella ornithinolytica in a 53-year-old-man [cited 2017 Jul 03]. Journal of Medical Microbiology 2014;1(3). Available from: http://jmmcr.microbiologyresearch.org/content/journal/jmmcr/10.1099/jmmcr.0.002634.
http://jmmcr.microbiologyresearch.org/co...
), enteric fever (Morais et al., 2009Morais VP, Daporta MT, Bao AF, Campello MG. Enteric fever-like syndrome caused by Raoultella ornithinolytica (Klebsiella ornithinolytica). Journal of Clinical Microbiology 2009;47(3):868-869.), biliary infection (Cleveland et al., 2014Cleveland KO, Mazumder SA, Gelfand MS. Association of Raoultella bacteremia with diseases of the biliary tract. Scandinavian Journal of Infectious Diseases 2014;46(7):541-542.; de Jong et al., 2014), infected “diabetic foot” (Solak et al., 2011Solak Y, Gul EE, Atalay H, Genc N, Tonbul HZ. A rare human infection of Raoultella ornithinolytica in a diabetic foot lesion. Annals of Saudi Medicine 2011;31(1):93-94.; Kabbara & Zgheib, 2015Kabbara WK, Zgheib YR. Diabetic foot infection caused by Raoultella ornithinolytica. American Journal of Health-System Pharmacy 2015;72(24):2147-2149.) and artritis (Venus et al.,2016Venus K, Vaithilingam S, Bogoch II. Septic arthritis of the knee due to Raoultella ornithinolytica. Infection 2016;44(5):691-692.). However, cases of infection were always related to some factor of primary malignancy or immune deficiency (Chun et al., 2015Chun S, Yun JW, Huh HJ, Lee NY.Clinical characteristics of Raoultella ornithinolytica bacteremia. Infection 2015;43(1):59-64.; Boattini et al., 2016Boattini M, Almeida A, Cardoso C, Cruz CS, Machado C, Vesza Z, et al. Infections on the rise:Raoultella spp., clinical and microbiological findings from a retrospective study, 2010-2014. Infectious Disease 2016;48(1):87-91.).

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., 2016Sala A, Taddei S, Santospirito D, Sandri C, Magnone W, Cabassi CS. Antibiotic resistance in conjunctival and enteric bacterial flora in raptors housed in a zoological Garden. Veterinary Medicine and Science 2016:2(4):239-245.) and in a case of hepatitis in Ring-Neck (Gonzales-Lama & Lupiola-Gomez, 2007Gonzales-Lama Z, Lupiola-Gomez PA. Entero bactéria senhígado de Psittacuserithacus. Revista Canaria de lãs Ciências Veterinarias 2007;4/5:11-13.). 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. (2009Morais VP, Daporta MT, Bao AF, Campello MG. Enteric fever-like syndrome caused by Raoultella ornithinolytica (Klebsiella ornithinolytica). Journal of Clinical Microbiology 2009;47(3):868-869.), Mau & Ross (2010Mau N, Ross LA. Raoultella ornithinolytica bacteremia in an infant with visceral heterotaxy. Pediatric Infectious Disease Journal 2010;29(5):477-478.), García-Lozano et al. (2013García-Lozano T, Plá FJP, Aznar-Oroval E. Raoultella ornithinolytica in urinary tract infections. Clinical and microbiological study of a series of 4 oncologic patients. Medicina Clinica 2013;141(3):138-139.), Kaya et al. (2015Kaya S, Bayramoglu G, Sönmez M, Köksal I. Raoultella ornithinolytica causing fatal sepsis. Brazilian Journal of Infect Diseases 2015:19(2):230-231.), Sekowska et al. (2015Sekowska A, Dylewska K, Gospodarek E, Bogiel T. Catheter-related blood stream infection caused by Raoultella ornithinolytica. Folia Microbiologica 2015;60(6):493-495.), Ponce-Alonso et al. (2016Ponce-Alonso M, Rodríguez-Rojas L, del Campo R, Cantón R, Morosini M-I. Comparison of different methods for identification of species of the genus Raoultella: report of 11 cases of Raoultella causing bacteraemia and literature review. Clinical Microbiology and Infection 2016;22(3):252-257.) and Seng et al. (2016Seng P, Boushab BM, Ramain F, Gouriet F, Bruder N, Martin C, et al. Emerging role of Raoultella ornithinolytica in human infections: a series of cases and review of literature. International Journal of Infect Diseases 2016;45(2016):65-71.), 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., 2004Walckenaer E, Poirel L, Leflon-Guibout V, Nordmann P, Nicolas-Chanoine MH. Genetic and biochemical characterization of the chromosomal class A b-lactamases of Raoultella (formerly Klebsiella) planticola and Raoultella ornithinolytica. Antimicrobial Agents and Chemotherapy 2004;48(1):305-312.). The macrolide resistance observed in this study was also expected since, according to Ito et al. (2005Ito NMK, Miyaji CI, Lima EA, Okabayashi S. Antimicrobianos:usos preventivos e curativos na avicultura. In: Palermo Neto J, Spinosa HS, Górniak SL. Farmacologia aplicada à avicultura: boas práticas no manejo de Medicamentos. São Paulo: Roca; 2005. p.115-147.), this pharmacological group is mainly indicated for Gram-positive 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., 2016Ponce-Alonso M, Rodríguez-Rojas L, del Campo R, Cantón R, Morosini M-I. Comparison of different methods for identification of species of the genus Raoultella: report of 11 cases of Raoultella causing bacteraemia and literature review. Clinical Microbiology and Infection 2016;22(3):252-257.), 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.

REFERENCES

  • Al-Hulu SM, Al-Charrakh AL, Al-Saadi MAK. Isolation and characterization of Raoultella ornithinolytica from clinical specimens in Hilla city, Iraq. Medical Journal of Babylon 2009;7(4):42-47.
  • Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 1966;45(4):493-496.
  • Boattini M, Almeida A, Cardoso C, Cruz CS, Machado C, Vesza Z, et al. Infections on the rise:Raoultella spp., clinical and microbiological findings from a retrospective study, 2010-2014. Infectious Disease 2016;48(1):87-91.
  • Chun S, Yun JW, Huh HJ, Lee NY.Clinical characteristics of Raoultella ornithinolytica bacteremia. Infection 2015;43(1):59-64.
  • Cleveland KO, Mazumder SA, Gelfand MS. Association of Raoultella bacteremia with diseases of the biliary tract. Scandinavian Journal of Infectious Diseases 2014;46(7):541-542.
  • Drancourt M, Bollet C, Carta A, Rousselier P. Phylogenetic analyses of Klebsiella species delineate Klebsiella and Raoultella gen. nov., with description of Raoultella ornithinolytica comb. nov., Raoultella terrigena comb. nov. and Raoultella planticola comb. nov. International Journal of Systematic and Evolutionary Microbiology 2001;51:925-932.
  • De Jong E, De Jong AS, Smidts-van den Berg N, Rentenaar RJ. Differentiation of Raoultella ornithinolytica/ planticola and Klebsiella oxytoca clinical isolates by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Diagnostic Microbiology and Infection Diseases 2013;75(4):431-433.
  • De Jong E, Erkens-Hulshof S, Van der Velden LB, Voss A, Bosboom R, Hodiamont CJ, et al. Predominant association of Raoultella bacteremia with diseases of the biliary tract. Scandinavian Journal of Infectious Diseases 2014;46(2):141-143.
  • García-Lozano T, Plá FJP, Aznar-Oroval E. Raoultella ornithinolytica in urinary tract infections. Clinical and microbiological study of a series of 4 oncologic patients. Medicina Clinica 2013;141(3):138-139.
  • Gonzales-Lama Z, Lupiola-Gomez PA. Entero bactéria senhígado de Psittacuserithacus. Revista Canaria de lãs Ciências Veterinarias 2007;4/5:11-13.
  • Hadano Y, Tsukahara M, Ito K, Suzuki J, Kawamura I, Kurai H. Raoultella ornithinolytica bacteremia in cancer patients: report of three cases. Internal Medicine 2012;51(22):3193-3195.
  • Ito NMK, Miyaji CI, Lima EA, Okabayashi S. Antimicrobianos:usos preventivos e curativos na avicultura. In: Palermo Neto J, Spinosa HS, Górniak SL. Farmacologia aplicada à avicultura: boas práticas no manejo de Medicamentos. São Paulo: Roca; 2005. p.115-147.
  • Kabbara WK, Zgheib YR. Diabetic foot infection caused by Raoultella ornithinolytica. American Journal of Health-System Pharmacy 2015;72(24):2147-2149.
  • KankiM, Yoda T, Tsukamoto T, Shibata T. Klebsiella pneumoniae produce no histamine: Raoultella planticola and Raoultella ornithinolytica strains are histamine producers. Applied in Environmental Microbiology 2007;68:3462-3466.
  • Haruki Y, Hagiya H, Sakuma A, Murase T, Sugiyama T, Kondo S. Clinical characteristics of Raoultella ornithinolytica bacteremia: a case series and literature review. Journal of Infection and Chemotherapy 2014;20(9):589-591.
  • Kaya S, Bayramoglu G, Sönmez M, Köksal I. Raoultella ornithinolytica causing fatal sepsis. Brazilian Journal of Infect Diseases 2015:19(2):230-231.
  • Masashi K, Tomoko Y, Teizo T, Tadayoshi S. Klebsiella pneumoniae produces no histamine: Raoultella planticola and Raoultella ornithinolytica strains are histamine producers. Applied and Environmental Microbiology 2002;68(7):3462-3466.
  • Mau N, Ross LA. Raoultella ornithinolytica bacteremia in an infant with visceral heterotaxy. Pediatric Infectious Disease Journal 2010;29(5):477-478.
  • Morais VP, Daporta MT, Bao AF, Campello MG. Enteric fever-like syndrome caused by Raoultella ornithinolytica (Klebsiella ornithinolytica). Journal of Clinical Microbiology 2009;47(3):868-869.
  • Nakasone ES, Kaneshiro R, Min K, Tokeshi J. Emergence of Raoultella ornithinolytica on O'ahu: a case of community-acquire R. Ornithinolytica urinary tract infection. Hawai'i Journal of Medicine & Public Health 2015;74(5):174-175.
  • Park JS, Hong KH, Lee HJ, Choi SH, Song SH, Song KH, et al. Evaluation of three phenotypic identification systems for clinical isolates of Raoultella ornithinolytica. Journal of Medical Microbiology 2011;60(4):492-499.
  • Podschun R, Sievers D, Fischer A, Ullmann U. Serotypes, hemma-gglutinins, siderophore synthesis, and serum resistance of Klebsiella isolates causing human urinary tract infections. Journal of Infectious Diseases 1993;168(6):1415-1421.
  • Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens, epidemiology, taxonomy, typing methods, and pathogencity factors. Clinical Microbiology Reviews 1998;11(4):589-603.
  • Ponce-Alonso M, Rodríguez-Rojas L, del Campo R, Cantón R, Morosini M-I. Comparison of different methods for identification of species of the genus Raoultella: report of 11 cases of Raoultella causing bacteraemia and literature review. Clinical Microbiology and Infection 2016;22(3):252-257.
  • Sala A, Taddei S, Santospirito D, Sandri C, Magnone W, Cabassi CS. Antibiotic resistance in conjunctival and enteric bacterial flora in raptors housed in a zoological Garden. Veterinary Medicine and Science 2016:2(4):239-245.
  • Seng P, Boushab BM, Ramain F, Gouriet F, Bruder N, Martin C, et al. Emerging role of Raoultella ornithinolytica in human infections: a series of cases and review of literature. International Journal of Infect Diseases 2016;45(2016):65-71.
  • Sekowska A, Dylewska K, Gospodarek E, Bogiel T. Catheter-related blood stream infection caused by Raoultella ornithinolytica. Folia Microbiologica 2015;60(6):493-495.
  • Sibanda M. Primary peritonitis caused by Raoultella ornithinolytica in a 53-year-old-man [cited 2017 Jul 03]. Journal of Medical Microbiology 2014;1(3). Available from: http://jmmcr.microbiologyresearch.org/content/journal/jmmcr/10.1099/jmmcr.0.002634
    » http://jmmcr.microbiologyresearch.org/content/journal/jmmcr/10.1099/jmmcr.0.002634
  • Solak Y, Gul EE, Atalay H, Genc N, Tonbul HZ. A rare human infection of Raoultella ornithinolytica in a diabetic foot lesion. Annals of Saudi Medicine 2011;31(1):93-94.
  • Sueifan M, Moog V, Rau E, Eichenauer T. Sepsis caused by Raoultella ornithinolyticain an immunocompetent patient. Der Anaesthesist 2016;65(2):120-133.
  • Venus K, Vaithilingam S, Bogoch II. Septic arthritis of the knee due to Raoultella ornithinolytica. Infection 2016;44(5):691-692.
  • Walckenaer E, Poirel L, Leflon-Guibout V, Nordmann P, Nicolas-Chanoine MH. Genetic and biochemical characterization of the chromosomal class A b-lactamases of Raoultella (formerly Klebsiella) planticola and Raoultella ornithinolytica. Antimicrobial Agents and Chemotherapy 2004;48(1):305-312.

Publication Dates

  • Publication in this collection
    Apr-Jun 2018

History

  • Received
    04 Aug 2017
  • Accepted
    30 Oct 2017
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