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Revista de Microbiologia

versión impresa ISSN 0001-3714

Rev. Microbiol. v. 29 n. 3 São Paulo Sept. 1998 



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

Submitted: March 21, 1997; Returned to authors for corrections: February 02, 1998;
Approved: July 23, 1998




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




The Nonfermentative Gram-negative Bacilli (NFGNB) are groups of strictly aerobic bacteria which use carbohydrates through a nonfermenting via (13). The NFGNB act as opportunistic pathogens. Their infections are from endogenous or exogenous origin, depending on several factors such as: use of immunosupressant substances, abusive use of wide spectrum antimicrobial agents, prolonged surgical procedures and inadequate mechanical instrumentation (3, 5, 6, 7, 8, 11, 18).

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. The bacterial strains were taken from the culture collection of the medical microbiology laboratory of the Universidade Federal do Ceará. The strains had been obtained from several clinical materials and comprised NFGNB stored for 14 months (January 1995 to February 1996). The strains had been previously classified as"Gram-Negative nonfermentative bacilli", Acinetobacter sp, Pseudomonas sp. and Pseudomonas aeruginosa. Four hundred fifty six NFGNB strains were used in this study.

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 95oF (35oC) for 18 to 24 hours and kept at room temperature for aditional 24 hours.

Biochemical studies. The samples were inoculated in BHI broth and incubated for 24 hours at 95oF (35oC). A loop of this culture was inoculated in the media and tests, except in the gelatinase production test, whose cultures were spot-inoculated.

All these media and tests were incubated at 35oC for 24 hours and reincubated for another 24 hours at room temperature. The gelatinase production test was incubated at 22oC for 48 hours. The remaining tests were read after 24 and after 48 hours, in order to confirm the results.



From January 1995 to February 1996, 1834 Gram-Negative bacilli were isolated and identified at Universidade Federal do Ceará microbiology laboratory. Among these, 1364 (74.4%) belonged to the Enterobacteriaceae family, 456 (24.8%) were Nonfermentative Gram-Negative Bacilli and 14 (0.8%) were Haemophillus genus bacilli.

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.

0006i01.gif (37097 bytes)

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

0006i02.gif (52357 bytes)

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.



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




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 




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* Corresponding author. Mailing address: Rua Trinta de Junho, 75 – casa 14, Água Fria, CEP 60834-260, Fortaleza, CE, Brasil. Fax: (+5585) 243-9316

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