Frequency of Nonfermentative Gram-Negative Bacilli Isolated from Clinical Materials of Pacients at Universidade Federal do Ceará Hospital Complex - Brazil

FREQUENCY OF NONFERMENTATIVE GRAM-NEGATIVE BACILLI ISOLATED FROM CLINICAL MATERIALS OF PACIENTS AT UNIVERSIDADE FEDERAL DO CEARÁ HOSPITAL COMPLEX - BRAZIL

Cristiane Cunha Frota^*, José Luciano Bezerra Moreira

Laboratório de Microbiologia, Departamento de Patologia e Medicina Legal, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará, Brasil

Approved: July 23, 1998

ABSTRACT

Among one thousand eight hundred and thirty-four Gram-negative bacilli, isolated at Universidade Federal do Ceará hospital complex – Brazil, from January 1995 to February 1996, 456 (24.8%) were Nonfermentative Gram-Negative Bacilli (NFGNB). This study reports their identification to the species level and their frequency as well. Thirteen genera and thirty species were identified and Pseudomonas aeruginosa was the most frequent species (69.95%), followed by Acinetobacter baumannii (5.48%) and by Acinetobacter lwoffii (3.95%). Among the identified P.aeruginosa strains, 94.1% produced pigment but 7.9% of them produced pigment only after being cultivated several times. The frequency of the most species was similar to that reported in the literature.

Key words: Nonfermentative Gram-Negative Bacilli, Pseudomonas aeruginosa

Until short time ago, the NFGNB were considered as commensals with little clinical importance. However, recent studies have shown that almost 15% (4, 9, 13, 14, 19) of all the isolations carried out by a routine clinical microbiology laboratory correspond to this group of bacteria.

In the laboratory, the occurrence of NFGNB is suspected by their reaction in the TSI (Triple Sugar Iron agar) medium, which is used in the identification of Gram-negative bacilli (20). The confirmation of a NFGNB can be obtained with precision through the use of the Hugh and Leifson O/F (Oxidation and Fermentation) medium (1, 13).

The purpose of this prospective study was to identify at the species level and to evaluate the frequency of the NFGNB isolated at Universidade Federal do Ceará hospital complex – Brazil, from January 1995 to February 1996.

Bacteria."Gram-Negative nonfermentative bacilli", Acinetobacter , Pseudomonas . Pseudomonas aeruginosa

Strains Maintenance. The strains were kept at room temperature in storage agar, in hermetically closed tubes. The tubes were sealed with rubber caps and kept away from light during the experiments. The stored cultures were renewed every two months.

Identification. The samples were identified using methods published by the American Society for Microbiology and adopted by Gillardi, 1991 (10) and Pickett et al., 1991 (19). Two hundred and eighty-three of the 456 strains of NFGNB were identified as P. aeruginosa based on presented the classical characteristics of the species, such as production of sweet odor, of pyocyanin and/or of pyoverdin, oxidase test positive reaction, positive motility and glucose acidification.

To check hemolysis cultures were inoculated in Columbia agar (Difco) containing 5% defibrinated sheep’s blood. The morphology and the pigmentation of the colonies, were studied using Iso-Sensitest agar (Oxoid).

The growth in brain heart infusion (BHI [Difco]) was used for preparing the smears for Gram’s staining, for performing the motility test and for fast growth of the cultures. All the cultures were incubated at 95

^oo

Biochemical studies. The samples were inoculated in BHI broth and incubated for 24 hours at 95

^oo

All these media and tests were incubated at 35

^ooEnterobacteriaceae, Haemophillus.

The distribution of NFGNB, according to the clinical source, is shown on Table 1. Most of the strains were isolated from urine (24.12 %) and blood (17.76 %).

Table 1.
Origin of the 456 Non Fermentative Gram-Negative Bacilli strains, isolated from clinical materials of patients from Universidade Federal do Ceará - Brazil hospital complex, 1995-1996.

Source: Universidade Federal do Ceará / Faculdade de Medicina / Department of Pathology and Legal Medicine / Microbiology Laboratory.

The 456 NFGNB strains studied belonged to 13 genera and 30 species. Among them, four (0.87%) could not be identified by the employed method. The most frequently isolated genus was Pseudomonas (346 strains – 75.87%), followed by Acinetobacter (48 strains - 10.52%). Pseudomonas aeruginosa species presented the highest frequency (69.95%), followed by Acinetobacter baumannii (5.48%), Acinetobacter lwoffii (3.95%) and Flavobacterium indologenes (3.51%). The Pseudomonas aeruginosa species remained as the main opportunist pathogen. The frequency of the species and the predominance of P. aeruginosa over the remaining NFGNB can be observed, in Table 2.

Table 2.
Identification of NFGNB strains, isolated from clinical materials of patients from Universidade Federal do Ceará - Brazil hospital complex, 1995-1996

Source: Universidade Federal do Ceará / Faculdade de Medicina / Department of Pathology and Legal Medicine / Microbiology Laboratory.

Among the 319 samples of P. aeruginosa, 300 (94.1%) produced pigments and, among these, 24 (7.9%) produced pigments at least in four subcultures. Among the pigment producing strains, 291 (97%) produced pyocyanin and 9 (3%) produced other pigments, such as pyorubin-pyomelanin (6 samples) and pyoverdin (3 samples).

The frequency of NFGNB (24.8%) was lower than that described in literature by Langle et al. (14), Motti and Neto (17), Romero et al. (21) and by Mimica and Mimica (16). Nevertheless, Pseudomonas aeruginosa remained the most frequent species (69.9%), with a frequency similar to those reported in other studies (2, 16, 21). The frequency of a few species, such as Acinetobacter baumannii (5.48%) and Xanthomonas (Stenotrophomonas) maltophilia (1.09%), were lower than the ones described by Motti et al. (18). We found a slightly higher frequency for Flavobacterium indologenes (3.75%) than that one published by Kitch et al. (2,5%) (12) and also for Flavimonas oryzihabitans (1.97%), when compared to the frequency reported by Pickett et al. (0.8%) (19). However, most of the identified species presented frequencies similar to the ones found by Martin et al. (15), Langle et al. (14) and Kitch et al. (12).

This study confirms that the group of NFGNB opportunist bacteria is not formed only by Pseudomonas aeruginosa but also by several others species. The identification of them provides important information to hospital infection control comissions, and leads doctors to a more suitable therapeutic conduct.

ACKNOWLEDGEMENTS

Technicians at Universidade Federal do Ceará microbiology laboratory and to CAPES.

RESUMO

Bacilos Gram-negativos não fermentadores isolados de materiais clínicos de pacientes do complexo hospitalar da Universidade Federal do Ceará - Brasil

Entre 1834 bacilos Gram-Negativos isolados no complexo hospitalar da Universidade Federal do Ceará – Brasil no período de janeiro de 1995 à fevereiro de 1996, 456 (24,8%) foram Bacilos Gram-Negativos Não Fermentadores (BGNNF). Este estudo relata sua identificação a nível de espécie e sua freqüência. Treze gêneros e trinta espécies foram identificadas, sendo que Pseudomonas aeruginosa foi a espécie mais freqüente (69,95%) seguida por Acinetobacter baumannii (5,48%) e Acinetobacter lwoffii (3,95%). Entre as cepas identificadas como P. aeruginosa, 94,1 % foram produtoras de pigmentos, mas 7,9% delas produziram pigmento somente após diversos subcultivos. A freqüência da maioria das espécies identificadas foi similar à relatada na literatura.

Palavras-chave: Bacilos Gram-negativos não fermentadores, Pseudomonas aeruginosa

REFERENCES

InBailey & Scott’s Diagnostic Microbiology^th

2. Berger U.; Piotrowski, H.D. The Identification Gram-negative bacteria. Experience with 676 apycyonagenic strains. Zentralb Bakteriol, Feb, 248:, 509-254, 1981.

3. Bermudez, L.E.M.; Dias, L.M.P. Infecções relacionadas a cateteres venosos profundos em pacientes com câncer: estudo bacteriológico. Folha Med., 93: 303-306, 1986.

4. Bouvet, P.J.M.; Grimont, P.AD. Identification and Biotyping of Clinical Isolates of Acinetobacter. Ann. Inst. Pasteur/ Microbiol., 138: 569-578, 1987.

5. Craven, D.E.; Moody, B.; Connoly, M.G.; Kollisch, N.R; Stottmeier, K.D; McCabe, W.R. Pseudobacteremia caused by Povidone-Iodine Solution Contaminated with Pseudomonas cepacia. N. Engl. J. Med., 305: 621-623, 1981

6. Dance, D.A.B. Meliodosis. Culture., 15: 2-4, 1994.

7. Faden, H.; Harabuchi, Y.; Hong, J.J.; et al. Epidemiology of Moraxella catarrhalis in Children during the First 2 Years of Life: Relationship to Otitis Media. J. Infect. Dis., 169: 1312-1317, 1994.

8. Fergie, J.E.; Shema, S.J.; Lott, L.; Crawford, R.; Patrick, C. Pseudomonas aeruginosa Bacteremia in Immunocompromised Children: Analysis of Factors Associated with a Poor Outcome. Clin. Infect. Dis, 18: 390-394, 1994.

9. Gerner-Smidt, P.; Tjernberg, I.; Ursing, J. Reliability of Phenotypic Tests for Identification of Acinetobacter Species. J. Clin. Microbiol, 29: 277-282, 1991.

10. Gilardi, G.L. Pseudomonas and Related Genera. In: Ballows, E.W.; Hausler, W.J.; Herrmann, K.L.; et al (eds). Manual of Clinical Microbiology. 5^th ed. American Society for Microbiology,Washington, D.C., 1991, p.429-441.

11. Holmes, B.; Popoff, M.; Kiredjan, M.; Kersters, K. Ochrobactrum anthropi gen. nov., sp. nov. from Human Clinical Specimens and Previously Known as Group Vd. Int. J. System. Bacteriol, 38: 406-416, 1988.

12. Kitch, T.T.; Jacobs, M.R.; Appelbaum, P.C. Evaluation of the 4-hour RapID Plus Method for Identification of 345 Gram-negative nonfermentative rods. J. Clin. Microbiol, 30: 1267-70, 1992.

13. Koneman, E.W.; Allen, S.D.; Janda,W.M.; et al. The Nonfermentative Gram-Negative Bacilli. In: Color Atlas and Textbook of Diagnostic Microbiology. 4^rd ed. J. B. Lippincott Company, Philadelphia, 1992, p. 185-242.

14. Langle, E.A.; Albores, P.S.; Perez, R.C.; Capellini, I.G. Métodos para Identificar Bacilos Gram-Negativos Non Fermentadores (BGNnF). Infectologia., 7: 287-298, 1987.

15. Martin, R.; Siavoshi, F.; McDougal, D.L. Comparison of Rapid NFT system and conventional methods for identification of nonsaccharolytic Gram-Negative Bacteria. J. Clin. Microbiol., 24:1089-92, 1986.

16. Mimica, I.; Mimica, L.N.J. Aztreonam, atividade in vitro frente a bactérias Gram-negativas. Arq. Bras. Med., 65:603-604, 1991.

17. Motti, E.F.; Neto, V.A. Padrões de resistência a antimicrobianos em bacilos Gra-negativos isolados de pacientes em unidade de terapia intensiva. Rer. Hosp. Clin. Fac. Med. S. Paulo, 47: 131-137, 1992.

18. Oto, M. A.; Fernandez, A.; Jerez, R.; Urriola, G.J. Acinetobacter en una Unidad Metropolitana de Neonatología: Aspectos Clínicos y Microbiológicos. Rev. Chil. Pediatr., 62:118-120, 1991.

19. Pickett, M.J.; Hollis, D.G.; Bottone, E.J. Miscellaneous Gram-Negative Bacteria, In: Ballows, E.W.; Hausler, W.J.; Herrmann, K.L.; et al. Manual of Clinical Microbiology. 5^th ed. American Society for Microbiology, Washington, D.C. 1991, p.410-428.

20. Pickett, M.J.; Pedersen, M.M. Nonfermentative Bacilli Associated with Man: II. Detection and Identification. Am. J. Clin. Pathol., 54:164-177, 1970.

21. Romero, L.E.; Monrás, M.F.P.; González, D.P.R.; Silva, J.L.Z. Identificacion de bactérias Gram-negativas no fermentadoras. Hospital pediatrico docente "Centro Habana". Dicienbre de 1996 a mayo de 1987. Rer. Cub. Med. Trop., 41: 274-283, 1989.

• ¹
  Baron, E.J.; Finegold, S.M. Nonfermentative Gram-Negative Bacilli and Coccobacilli. In: Bailey & Scott’s Diagnostic Microbiology 8^th ed. The C.V. Mosby Company, St. Louis Missouri, 1990, p. 386-406.
• ²
  Berger U.; Piotrowski, H.D. The Identification Gram-negative bacteria. Experience with 676 apycyonagenic strains. Zentralb Bakteriol, Feb, 248:, 509-254, 1981.
• ³
  Bermudez, L.E.M.; Dias, L.M.P. Infecções relacionadas a cateteres venosos profundos em pacientes com câncer: estudo bacteriológico. Folha Med, 93: 303-306, 1986.
• ⁴
  Bouvet, P.J.M.; Grimont, P.AD. Identification and Biotyping of Clinical Isolates of Acinetobacter Ann. Inst. Pasteur/ Microbiol, 138: 569-578, 1987.
• ⁵
  Craven, D.E.; Moody, B.; Connoly, M.G.; Kollisch, N.R; Stottmeier, K.D; McCabe, W.R. Pseudobacteremia caused by Povidone-Iodine Solution Contaminated with Pseudomonas cepacia. N. Engl. J. Med., 305: 621-623, 1981
• ⁶
  Dance, D.A.B. Meliodosis. Culture., 15: 2-4, 1994.
• ⁷
  Faden, H.; Harabuchi, Y.; Hong, J.J.; et al. Epidemiology of Moraxella catarrhalis in Children during the First 2 Years of Life: Relationship to Otitis Media. J. Infect. Dis., 169: 1312-1317, 1994.
• ⁸
  Fergie, J.E.; Shema, S.J.; Lott, L.; Crawford, R.; Patrick, C. Pseudomonas aeruginosa Bacteremia in Immunocompromised Children: Analysis of Factors Associated with a Poor Outcome. Clin. Infect. Dis, 18: 390-394, 1994.
• ⁹
  Gerner-Smidt, P.; Tjernberg, I.; Ursing, J. Reliability of Phenotypic Tests for Identification of Acinetobacter Species. J. Clin. Microbiol, 29: 277-282, 1991.
• ¹⁰
  Gilardi, G.L. Pseudomonas and Related Genera. In: Ballows, E.W.; Hausler, W.J.; Herrmann, K.L.; et al (eds). Manual of Clinical Microbiology. 5^th ed. American Society for Microbiology,Washington, D.C., 1991, p.429-441.
• ¹¹
  Holmes, B.; Popoff, M.; Kiredjan, M.; Kersters, K. Ochrobactrum anthropi gen. nov., sp. nov. from Human Clinical Specimens and Previously Known as Group Vd. Int. J. System. Bacteriol, 38: 406-416, 1988.
• ¹²
  Kitch, T.T.; Jacobs, M.R.; Appelbaum, P.C. Evaluation of the 4-hour RapID Plus Method for Identification of 345 Gram-negative nonfermentative rods. J. Clin. Microbiol, 30: 1267-70, 1992.
• ¹³
  Koneman, E.W.; Allen, S.D.; Janda,W.M.; et al. The Nonfermentative Gram-Negative Bacilli. In: Color Atlas and Textbook of Diagnostic Microbiology. 4^rd ed. J. B. Lippincott Company, Philadelphia, 1992, p. 185-242.
• ¹⁴
  Langle, E.A.; Albores, P.S.; Perez, R.C.; Capellini, I.G. Métodos para Identificar Bacilos Gram-Negativos Non Fermentadores (BGNnF). Infectologia., 7: 287-298, 1987.
• ¹⁵
  Martin, R.; Siavoshi, F.; McDougal, D.L. Comparison of Rapid NFT system and conventional methods for identification of nonsaccharolytic Gram-Negative Bacteria. J. Clin. Microbiol., 24:1089-92, 1986.
• ¹⁶
  Mimica, I.; Mimica, L.N.J. Aztreonam, atividade in vitro frente a bactérias Gram-negativas. Arq. Bras. Med., 65:603-604, 1991.
• ¹⁷
  Motti, E.F.; Neto, V.A. Padrões de resistência a antimicrobianos em bacilos Gra-negativos isolados de pacientes em unidade de terapia intensiva. Rer. Hosp. Clin. Fac. Med. S. Paulo, 47: 131-137, 1992.
• ¹⁸
  Oto, M. A.; Fernandez, A.; Jerez, R.; Urriola, G.J. Acinetobacter en una Unidad Metropolitana de Neonatología: Aspectos Clínicos y Microbiológicos. Rev. Chil. Pediatr., 62:118-120, 1991.
• ¹⁹
  Pickett, M.J.; Hollis, D.G.; Bottone, E.J. Miscellaneous Gram-Negative Bacteria, In: Ballows, E.W.; Hausler, W.J.; Herrmann, K.L.; et al. Manual of Clinical Microbiology. 5^th ed. American Society for Microbiology, Washington, D.C. 1991, p.410-428.
• ²⁰
  Pickett, M.J.; Pedersen, M.M. Nonfermentative Bacilli Associated with Man: II. Detection and Identification. Am. J. Clin. Pathol., 54:164-177, 1970.
• ²¹
  Romero, L.E.; Monrás, M.F.P.; González, D.P.R.; Silva, J.L.Z. Identificacion de bactérias Gram-negativas no fermentadoras. Hospital pediatrico docente "Centro Habana". Dicienbre de 1996 a mayo de 1987. Rer. Cub. Med. Trop., 41: 274-283, 1989.

*

Corresponding author. Mailing address: Rua Trinta de Junho, 75 – casa 14, Água Fria, CEP 60834-260, Fortaleza, CE, Brasil. Fax: [phone]

Publication Dates

• Publication in this collection
  26 Feb 1999
• Date of issue
  Sept 1998

History

• Accepted
  23 July 1998
• Reviewed
  02 Feb 1998
• Received
  21 Mar 1997