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Chapter 31 : Approaches to the Identification of Aerobic Gram-Negative Bacteria

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Abstract:

This chapter describes the approach to the identification of gram-negative rods, with emphasis on the greater difficulty in identifying non-glucose-fermenting organisms. Before presenting the identification scheme, several issues of terminology and methodology are addressed. The chapter uses more precise terminology for the terms utilization, oxidation, and glycolysis. Careful attention to testing methods is critical when describing their application for nonfermenters. Slight variations of reagents or incubation conditions can cause numerous mis-identifications and have caused erroneous descriptions of new species. The chapter provides limited description of assimilation assays for the identification of nonfermenters to that of acetamide and acetate, used solely as additional characteristics to differentiate between some species. Arginine dihydrolase activity by nonfermenters can best be tested by dense inoculation in Moeller broth with 1% arginine. Susceptibility to desferrioxamine, an Fe chelator, can be tested on Mueller-Hinton agar (MHA), or on tryptic soy agar (TSA) for strains growing poorly on MHA, using paper disks loaded with 250 µg of desferrioxamine or using commercially available Rosco tablets. Trypsin or benzyl-arginine arylamidase activity is very discriminative for nonfermenters, since the strains of almost all taxa exhibit an all-or-nothing positive result, providing a test with high discriminatory value. Some gram-negative organisms are vancomycin susceptible, and this can be used as a rapid and distinctive identification tool.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31

Key Concept Ranking

Antibacterial Agents
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Sugar Alcohols
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Gram-Negative Bacteria
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Carbohydrates
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Gram-Positive Bacteria
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Nitrate Reductase
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References

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1. Bernardet, J.-F.,, Y. Nakagawa,, and B. Holmes. 2002. Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int. J. Syst. Evol. Microbiol. 52:10491070.
2. Bombicino, K.A.,, M. N. Almuzara,, A. M. Famiglietti,, and V. Vay. 2007. Evaluation of pyrrolidonyl arylamidase for the identification of nonfermenting Gram-negative rods. Diagn. Microbiol. Infect. Dis. 57:101103. (Erratum, 57:473.)
3. Coenye, T.,, E. Falsen,, M. Vancanneyt,, B. Hoste,, J. R. W. Govan,, K. Kersters,, and P. Vandamme. 1999. Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov. Int. J. Syst. Bacteriol. 49:405413.
4. Coenye, T.,, J. Goris,, P. De Vos,, P. Vandamme,, and J. J. LiPuma. 2003. Classification of Ralstonia pickettii-like isolates from the environment and clinical samples as Ralstonia insidiosa sp. nov. Int. J. Syst. Evol. Microbiol. 53:10751080.
5. Cruze, J. A.,, J. T. Singer,, and W. R. Finnerty. 1979. Conditions for quantitative transformation in Acinetobacter calcoaceticus. Curr. Microbiol. 3:129132.
6. De Backer, E.,, R. Verhelst,, H. Verstraelen,, M. A. Alqumber,, J. P. Burton,, J. R. Tagg,, M. Temmerman,, and M. Vaneechoutte. 2007. Quantitative determination by realtime PCR of four vaginal Lactobacillus species, Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners. BMC Microbiol. 7:115.
7. Holmes, B.,, R. J. Owen,, A. G. Steigerwalt,, and D. J. Brenner. 1984. Flavobacterium gleum, a new species found in human clinical specimens. Int. J. Syst. Bacteriol. 34:2125.
8. Hugh, R.,, and E. Leifson. 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. J. Bacteriol. 66:2426.
9. Kämpfer, P.,, M. Vaneechoutte,, N. Lodders,, T. De Baere,, V. Avesani,, M. Janssens,, H.-J. Busse,, and G. Wauters. 2009. Description of Chryseobacterium anthropi sp. nov., to accommodate clinical isolates biochemically similar to Kaistella koreensis and Chryseobacterium haifense, proposal to reclassify Kaistella koreensis as Chryseobacterium koreense comb. nov. and emended description of the genus Chryseobacterium. Int. J. Syst. Evol. Microbiol. 59:24212428.
10. Kodaka, H.,, A. Y. Armfield,, G. L. Lombard,, and V. R. Dowell, Jr. 1982. Practical procedure for demonstrating bacterial flagella. J. Clin. Microbiol. 16:948952.
11. Laffineur, K.,, M. Janssens,, J. Charlier,, V. Avesani,, G. Wauters,, and M. Delmée. 2002. Biochemical and susceptibility tests useful for identification of nonfermenting gramnegative rods. J. Clin. Microbiol. 40:10851087.
12. Martin, R.,, P. S. Riley,, D. G. Hollis,, R. E. Weaver,, and M. I. Krichevsky. 1981. Characterization of some groups of gram-negative nonfermentative bacteria by the carbon source alkalinization technique. J. Clin. Microbiol. 14:3947.
13. Quan, Z.-X.,, K. K. Kim,, M.-K. Kim,, L. Jin,, and S.-T. Lee. 2007. Chryseobacterium caeni sp. nov., isolated from bioreactor sludge. Int. J. Syst. Evol. Microbiol. 57:141145.
14. Vaneechoutte, M.,, P. Kämpfer,, T. De Baere,, E. Falsen,, and G. Verschraegen. 2004. Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov. Int. J. Syst. Evol. Microbiol. 54:317327.
15. Wauters, G.,, B. Van Bosterhaut,, M. Janssens,, and J. Verhaegen. 1998. Identification of Corynebacterium amycolatum and other nonlipophilic fermentative corynebacteria of human origin. J. Clin. Microbiol. 36:14301432.
16. Yabuuchi, E.,, T. Kaneko,, I. Yano,, C. W. Moss,, and N. Miyoshi. 1983. Sphingobacterium gen. nov., Sphingobacterium spiritivorum comb. nov., Sphingobacterium multivorum com. nov., Sphingobacterium mizutae sp. nov., and Flavobacterium indologenes sp. nov.: glucose-nonfermenting gram-negative rods in CDC groups IIk-2 and IIb. Int. J. Syst. Bacteriol. 33:580598.

Tables

Generic image for table
TABLE 1

Dichotomous identification algorithm for gram-negative bacteria with poor or no growth on SBA

p, positive; pp, strongly (quickly) positive; (p), delayed positive; p, weakly positive; p/(p), positive or delayed positive; p/p, positive or weakly positive; n, negative; → [number], go to number indicated further on in the table. Chapter numbers in parentheses indicate the chapters in which the organism(s) is described further in this .

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 4

Dichotomous identification algorithm for aerobically growing gram-negative nonfermenters (strict aerobes)

Nonfermenters are usually referred to as strict aerobes, although some nonfermenters, such as all denitrifiers (producing nitrogen gas from nitrite), can grow anaerobically when nitrate and/or nitrite is available as an alternative electron acceptor in oxidative respiration, instead of gaseous oxygen. p, positive; pp, strongly (quickly) positive; (p), delayed positive; p, weakly positive; p/(p), positive or delayed positive; p/p, positive or weakly positive; n, negative; → [number], go to number indicated further on in the table. Chapter numbers in parentheses indicate the chapters in which the organism(s) is described further in this Manual. partim, Latin term used to indicate “the part that is.”

, , and are 100%, and 95%, and 65% pyoverdin production p.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 2

Dichotomous identification algorithm for aerobically growing gram-negative bacteria which form purple or pink colonies on SBA

p, positive; pp, strongly (quickly) positive; (p), delayed positive; p, weakly positive; p/(p), positive or delayed positive; p/p, positive or weakly positive; n, negative; → [number], go to number indicated further on in the table. Chapter numbers in parentheses indicate the chapters in which the organism(s) is described further in this .

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 5a

Overview of biochemical characteristics of the gram-negative nonfermenters

Characteristics useful for identification according to the dichotomous Table 4 are listed in boldface type; the distinction between groups in each dichotomy is indicated by roman versus italic type. Alphanumeric data are percentages of positive isolates. MCA, MacConkey agar; n, negative; n*, negative, but some isolates of this species are positive for this characteristic, dealt with elsewhere in the table; n/p, most strains negative; o, data not listed; p, positive; pp, strongly positive; p*, positive, but some isolates of this species are negative for this characteristic, dealt with elsewhere in the table; p, weakly positive; (p), delayed positive; (p), delayed and weakly positive; p/(p), most strains positive, some delayed positive; p/n, most strains positive; p/p, most strains positive, some weakly positive; R, resistant; R/S, most strains resistant; S, susceptible; sheep blood agar; S/R: most strains susceptible; S [number], percentage of susceptible strains; v, strain dependent.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 5b

Overview of biochemical characteristics of the gram-negative nonfermenters

Characteristics useful for identification according to the dichotomous Table 4 are listed in boldface type; the distinction between groups in each dichotomy is indicated by roman versus italic type. Alphanumeric data are percentages of positive isolates. MCA, MacConkey agar; n, negative; n*, negative, but some isolates of this species are positive for this characteristic, dealt with elsewhere in the table; n/p, most strains negative; o, data not listed; p, positive; pp, strongly positive; p*, positive, but some isolates of this species are negative for this characteristic, dealt with elsewhere in the table; p, weakly positive; (p), delayed positive; (p), delayed and weakly positive; p/(p), most strains positive, some delayed positive; p/n, most strains positive; p/p, most strains positive, some weakly positive; R, resistant; R/S, most strains resistant; S, susceptible; sheep blood agar; S/R: most strains susceptible; S [number], percentage of susceptible strains; v, strain dependent.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 5c

Overview of biochemical characteristics of the gram-negative nonfermenters

Characteristics useful for identification according to the dichotomous Table 4 are listed in boldface type; the distinction between groups in each dichotomy is indicated by roman versus italic type. Alphanumeric data are percentages of positive isolates. MCA, MacConkey agar; n, negative; n*, negative, but some isolates of this species are positive for this characteristic, dealt with elsewhere in the table; n/p, most strains negative; o, data not listed; p, positive; pp, strongly positive; p*, positive, but some isolates of this species are negative for this characteristic, dealt with elsewhere in the table; p, weakly positive; (p), delayed positive; (p), delayed and weakly positive; p/(p), most strains positive, some delayed positive; p/n, most strains positive; p/p, most strains positive, some weakly positive; R, resistant; R/S, most strains resistant; S, susceptible; sheep blood agar; S/R: most strains susceptible; S [number], percentage of susceptible strains; v, strain dependent.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 5d

Overview of biochemical characteristics of the gram-negative nonfermenters

Characteristics useful for identification according to the dichotomous Table 4 are listed in boldface type; the distinction between groups in each dichotomy is indicated by roman versus italic type. Alphanumeric data are percentages of positive isolates. MCA, MacConkey agar; n, negative; n*, negative, but some isolates of this species are positive for this characteristic, dealt with elsewhere in the table; n/p, most strains negative; o, data not listed; p, positive; pp, strongly positive; p*, positive, but some isolates of this species are negative for this characteristic, dealt with elsewhere in the table; p, weakly positive; (p), delayed positive; (p), delayed and weakly positive; p/(p), most strains positive, some delayed positive; p/n, most strains positive; p/p, most strains positive, some weakly positive; R, resistant; R/S, most strains resistant; S, susceptible; sheep blood agar; S/R: most strains susceptible; S [number], percentage of susceptible strains; v, strain dependent.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 5e

Overview of biochemical characteristics of the gram-negative nonfermenters

Characteristics useful for identification according to the dichotomous Table 4 are listed in boldface type; the distinction between groups in each dichotomy is indicated by roman versus italic type. Alphanumeric data are percentages of positive isolates. MCA, MacConkey agar; n, negative; n*, negative, but some isolates of this species are positive for this characteristic, dealt with elsewhere in the table; n/p, most strains negative; o, data not listed; p, positive; pp, strongly positive; p*, positive, but some isolates of this species are negative for this characteristic, dealt with elsewhere in the table; p, weakly positive; (p), delayed positive; (p), delayed and weakly positive; p/(p), most strains positive, some delayed positive; p/n, most strains positive; p/p, most strains positive, some weakly positive; R, resistant; R/S, most strains resistant; S, susceptible; sheep blood agar; S/R: most strains susceptible; S [number], percentage of susceptible strains; v, strain dependent.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 5f

Overview of biochemical characteristics of the gram-negative nonfermenters

Characteristics useful for identification according to the dichotomous Table 4 are listed in boldface type; the distinction between groups in each dichotomy is indicated by roman versus italic type. Alphanumeric data are percentages of positive isolates. MCA, MacConkey agar; n, negative; n*, negative, but some isolates of this species are positive for this characteristic, dealt with elsewhere in the table; n/p, most strains negative; o, data not listed; p, positive; pp, strongly positive; p*, positive, but some isolates of this species are negative for this characteristic, dealt with elsewhere in the table; p, weakly positive; (p), delayed positive; (p), delayed and weakly positive; p/(p), most strains positive, some delayed positive; p/n, most strains positive; p/p, most strains positive, some weakly positive; R, resistant; R/S, most strains resistant; S, susceptible; sheep blood agar; S/R: most strains susceptible; S [number], percentage of susceptible strains; v, strain dependent.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 5g

Overview of biochemical characteristics of the gram-negative nonfermenters

Characteristics useful for identification according to the dichotomous Table 4 are listed in boldface type; the distinction between groups in each dichotomy is indicated by roman versus italic type. Alphanumeric data are percentages of positive isolates. MCA, MacConkey agar; n, negative; n*, negative, but some isolates of this species are positive for this characteristic, dealt with elsewhere in the table; n/p, most strains negative; o, data not listed; p, positive; pp, strongly positive; p*, positive, but some isolates of this species are negative for this characteristic, dealt with elsewhere in the table; p, weakly positive; (p), delayed positive; (p), delayed and weakly positive; p/(p), most strains positive, some delayed positive; p/n, most strains positive; p/p, most strains positive, some weakly positive; R, resistant; R/S, most strains resistant; S, susceptible; sheep blood agar; S/R: most strains susceptible; S [number], percentage of susceptible strains; v, strain dependent.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 5h

Overview of biochemical characteristics of the gram-negative nonfermenters

Characteristics useful for identification according to the dichotomous Table 4 are listed in boldface type; the distinction between groups in each dichotomy is indicated by roman versus italic type. Alphanumeric data are percentages of positive isolates. MCA, MacConkey agar; n, negative; n*, negative, but some isolates of this species are positive for this characteristic, dealt with elsewhere in the table; n/p, most strains negative; o, data not listed; p, positive; pp, strongly positive; p*, positive, but some isolates of this species are negative for this characteristic, dealt with elsewhere in the table; p, weakly positive; (p), delayed positive; (p), delayed and weakly positive; p/(p), most strains positive, some delayed positive; p/n, most strains positive; p/p, most strains positive, some weakly positive; R, resistant; R/S, most strains resistant; S, susceptible; sheep blood agar; S/R: most strains susceptible; S [number], percentage of susceptible strains; v, strain dependent.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31
Generic image for table
TABLE 3

Dichotomous identification algorithm for aerobically growing gram-negative bacteria fermenting glucose (facultative anaerobes)

p, positive; pp, strongly (quickly) positive; (p), delayed positive; p, weakly positive; p/(p), positive or delayed positive; p/p, positive or weakly positive; n, negative; → number], go to number indicated further on in the table. Chapter numbers in parentheses indicate the chapters in which the organism(s) is described further in this Manual.

Citation: Wauters G, Vaneechoutte M. 2011. Approaches to the Identification of Aerobic Gram-Negative Bacteria , p 539-558. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch31

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