species

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A Comparison of Fluorescent Pigments FromFlavobacterium and Sphingobacterium SpeciesThomas M. Rossi a , C. Wayne Moss b & Isiah M. Warner ca Department of Chemistry Emory, University Atlanta, Georgia, 30322b Centers for Disease Control Building, 5/Room 308, Atlanta, Georgia, 30333c Smith Kline & French Laboratories F-70, 1500 Spring Garden Street, Philadelphia, PA, 19101Published online: 05 Dec 2006.

To cite this article: Thomas M. Rossi , C. Wayne Moss & Isiah M. Warner (1986) A Comparison of Fluorescent Pigments FromFlavobacterium and Sphingobacterium Species, Analytical Letters, 19:13-14, 1457-1486, DOI: 10.1080/00032718608069119

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ANALYTICAL LETTERS, 1 9 ( 1 3 & 1 4 ) , 1457-1486 ( 1 9 8 6 )

A COMPARISON OF FLUORESCENT PIGMENTS FROM FLAVOBACTERIUM AND SPHINGOBACTERIUM SPECIES

KEY WORDS: Fluorometry, identification of fluorescent pigments, bacteria, pigment production, microorganisms, "fingerprinting" organisms

Th0ma.s M. Rossi,+ C. Wayne Moss' and Isiah M. Warner*

* Department of Chemistry Emory University

Atlanta, Georgia 30322

# Centers for Disease Control Building !j/ROOm 308

Atlanta, Georgia 30333

-b Present Address: Smith Kline & French Laboratories F-70, 1500 Spring Garden Street

Philadelphia, PA 19101

ABSTRACT

An extraction procedure was developed for the isolation o f

fluorescent pigments from species of Flavobacterium and

- Sphingobacterium. --- ---___-__- This procedure was conducted at room

temperature. Thereby the possibility of thermal degradation of

the fluorescent pigments present in these organisms was reduced

compared to previously reported extraction methods in which

pigments were extracted after heating with strong base. Extracts

* To whom correspondence should be addressed

1457

Copyright 0 1986 by Marcel Dekker, Inc. 0003-2719/86/19 13-1457$3.50/0

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1458 ROSSI, MOSS, AND WARNER

from strains of eight species of these two genera have been

evaluated using a video fluorometer (VF). This instrument rapidly

provides a two-dimensional fluorescence spectrum of each extract.

Data from replicate extracts of the organisms have been compared in

order to evaluate the potential for identification of these

microorganisms on the basis of their fluorescent pigment content.

Similarities between the fluorescence spectra of two of the

Flavobacterium species and Sphingobacteriug mizutae support earlier

studies which have shown that these organisms are related on the

basis of their content of menaquinones and cellular fatty acids.

INTRODUCTION

Flavobacterium is literally translated from latin as a

"yellow, small rod," shaped bacterium1 . The original taxonomic

description of this genus was vaguG, so a variety of yellow

pigmented bacteria had been classified as belonging to the genus.

Recently, more well defined genus descriptors have resulted in the

reassignment of some gram positive and genetically distinct species

to other genera . However, efforts are continuing to further

define the genus.

2

Recently, the genus Sphingobacterium has been proposed for two

species of Flavobacterium (L multivorum and spiritovorum) .

Furthermore, a newly reported species ( L thalpophilum) was

recently found to have several similar characteristics to the

Sphingobacterium group . These three species have been found

to have high phosphosphingolipid contents as compared to other

Flavobacterium species. Since pigmentation is of historical

3

4

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COMPARISON OF FLUORESCENT PIGMENTS 1459

importance in the classification of Flavobacterium and since

fluorescent pigments have recently been found in some of the

organisms4 , the present study was conducted to evaluate the

potential for differentiation between Flavobacterium and

Sphingobacterium species by examining the profiles of fluorescent

pigments extracted from whole cells. In addition to providing a

means for differentiating between these organisms, the general

characteristics of the fluorescence spectra of the extracts from

these bacteria will be shown to be useful for evaluating the

similarities between Sphingobacterium and the Flavobacterium

species.

The first portion of the work reported in this study relates

the development of an extraction scheme for the isolati-on of

fluorescent pigments from various strains of Flavobacterium. The

extraction scheme used by Dees et al. in which fluorescent

pigments were reported in the extracts, was tested as a means of

isolating fluorescent pigments from all the Flavobacterium species

examined, but was found to be unsatisfactory for this purpose.

That extraction scheme was originally used for the isolation of

isoprenoid quinones from the bacterial cells, and hence, should not

be expected to be the optimum scheme for the recovery of

fluorescent pigments. In the present study, a number of solvents

have been tested for their utility in the extraction of fluorescent

components from suspensions of sonicated bacterial cells. The

optimized extraction method is shown to be capable of isolating a

variety of pigments with relatively high efficiency, and without

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heating the cells during the extraction process. Thus, the

probability of thermal decomposition of the pigments during sample

preparation is reduced.

In the second phase of this work, the optimized extraction

scheme was applied to the isolation of pigments from strains of

seven species of Flavobacterium and from strains of

Sphingobacterium mizutae. The extracts obtained from the

microorganisms have been evaluated using a video fluorometer (VF) . 5,6

The VF is a fluorescence spectrometer capable of acquiring

data in a two-dimensional matrix known as an excitation-emission

matrix (EEM). Each element of an EEM represents the intensity of

fluorescence of a sample monitored at a unique excitation and

emission wavelength pair. Due to the selectivity inherent in EEM

data, the VF has been found to be useful for the identification of

microorganisms based on both intrinsic fluorescence 7 'a and the

interaction of bacterial components with fluorescent dyes

A review of the utility of video fluorometry for the identification

of microorganisms has been recently published

9,lO

11

In addition to visual evaluation of the EEMs of the extracts

reported in this study, a pattern recognition techniquebased on 1 3 L L

the two-dimensional Fourier transform has been used to

demonstrate the automatic identification of the organisms studied

in the present work based on extract EEMs. Since the mathematical

basis of the pattern recognition algorithm used in this study has AI been previously reported , details concerning the algorithm

will not be presented here.

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MATERIALS AND METHODS

Apparatus

The video fluorometer used i n the present study was s i m i l a r t o

one r e p o r t e d p r e v i o u s l y i n t h e l i t e r a t u r e . Ins t rumen t

c o n t r o l and d a t a man ipu la t ion were conducted u s i n g a Hewle t t

Packard 9845B desktop minicomputer (Palo Alto, CA 94304.)

5,6,12

A r e u s a b l e s y r i n g e t i p f i l t e r ( M i l l i p o r e Co., Bedford, MA

07130) was used f o r removal of p a r t i c u l a t e m a t t e r from c e l l u l a r

e x t r a c t s . Hydrophobic f i l t e r s w i t h 0.5 r m pore s i z e were a l s o

purchased from Millipore.

Reagents

Ethanol (EtOH) was purchased from AAPER Alcohol and Chemical

Co. ( S h e l b y v i l l e , KY 40065). Dimethyl s u l f o x i d e (DMSO) and mixed

hexanes were purchased from Fischer Sc ien t i f i c Co. (Fairlawn, N J

07410) . E t h e r was o b t a i n e d f rom J . T. Bake r C h e m i c a l Co.

(Phill ipsburg, N J 08865). Phosphate buffered s a l i n e (FBS) (pH 7.0

- 7.2) was prepared a t the Centers fo r Disease Control (CDC).

Procedures

The s e v e n s p e c i e s o f F l a v o b a c t e r i u m and t h e s i n g l e

Sphingobacterium species used i n t h i s study a re l i s t e d i n Table I.

These organisms w e r e grown from CDC stock cu l tu re s on blood agar

p l a t e s f o r a p e r i o d of 48 h r s a t 35' C. A f t e r h a r v e s t i n g , t h e

organisms were heat k i l l e d , washed twice with d i s t i l l e d water and

then frozen u n t i l needed f o r analysis.

I n order t o develop an optimized extract ion procedure, thawed

samples of the seven species of Flavobacterium were mixed and then

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TABLE 1

List of Flavobacterium and Sphingobacterium Species Used in This Study.

Flavobacterium breve

Flavobac terium indologenes

Flavobacterium meningosepticum

Flavobacterium multivorum

Flavobacterium odoratum

Flavobacterium spiritovorum

Flavobacterium thalpophilum

Sphingobacterium mizutae

subdivided into five homogeneous subsamples (each subsample weighed

approximately 600mg). Each of the subsamples was extracted with

various solvent systems. A list of the solvents used in this

portion of the study is provided in Table 11. The general

extraction procedure followed is outlined below.

First, the cell sample was washed with approximately 8 ml of

PBS. After centrifugation and removal of the PBS, 1 ml of

extraction solvent was added to the cell pellet and the cells were

dispersed with vigorous vortexing. The cell suspension was then

sonicated for 10 minutes at room temperature by placing the test

tube containing the suspension in a water filled beaker which was

in turn placed in the sonicator. Sonication was used to disrupt

the cells without heating. After sonication, the cells were

separated from the extraction solvent by centrifugation. The

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TABLE 2

Solvents Tested for Extraction of Pigments from Sonicated Bacterial Cells.

Dimethylsulfoxide (DMSO)

Ethanol (EtOH)

Ether

Ether:Hexane 1:l (v/v)

Hexane

Note: Abbreviations given in parentheses are used throughout the manuscript.

solvent was then decanted and saved. The cell pellets were reex-

tracted following the identical procedure. Finally, the cell

pellets were extracted a third time. For the third extraction, 2

ml of solvent was used and the cells were allowed to sonicate for

15 min. The solvents from the three extractions were combined and

the total volume was approximately 4 ml.

The collected extracts were then filtered using a syringe type

teflon filter with changeable filter membranes using a fresh

membrane for each extract. Filtration was necessary to reduce

interferences from scattered light during fluorometric evaluation

of the extracts. The filtered extracts were examined directly

using the video fluorometer. The five solvents listed in Table I1

were compared for extractability of the fluorescent pigments from

the bacteria. The optimum solvents were then tested in combination

in order to further enhance the extraction efficiency.

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1464 ROSSI , MOSS, AND WARNER

Once a solvent system had been chosen, strains of each of the

eight species of bacteria listed in Table I were extracted

individually. The EEMs of these extracts were acquired using the

video fluorometer. The reproducibility of the extraction procedure

was investigated by extracting a second growth of the organisms and

comparing the EEMs of the extracts of these organisms to the EEMs

of the extracts from the first growth. In the case of 5 breve,

the replicate extract was taken from the first growth rather than

the second. This allowed for a check of the reproducibility of the

extraction procedure without introducing possible variability from

the growth procedure. Fourier transform based pattern recognition

was applied to these EEMs using one set of extracts as the

12

standard library and the second set as unknowns.

RESULTS AND DISCUSSION

Optimization of Extraction Procedure

Isometric projections and monochromatic images of the EEMs of

the cellular extracts obtained using each of the five test solvents

are shown in Figure 1 through 5. It is evident from these figures

that a complex set of pigments can be extracted from the organisms.

The hexane extract contained the lowest concentration of

fluorescent pigments, whereas the ethanol extract contained the

greatest variety and highest concentration of pigments. The ether

extract had a high concentration of a fluorescent pigment which was

blue shifted in excitation and emission relative to the pigments

contained in most of the other extracts. Hence, for the extraction

of this particular pigment, ether was the best solvent tested.

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n

E C

z 0

l-

I- V X w

v

H

a H

3913

4 7 0

45 1

432

4 13

394

37s

356

337

318

299 398 423 448 473 498 523 548 573 590 623

EMISSION ( n m )

FIG. 1. Isometric projection and monochromatic image of the EEM of an ether extract from a mixture o f Flavobacterium species.

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CI

E C

z I- + U X W

v

2 a H

470

45 1

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4 13

394

375

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3 10

299 390 423 448 473 498 523 548 573 598 623

EMISSION ( n m )

FIG. 2. Isometric projection and monochromatic image of a DMSO extract from a mixture of Flavobacterium species.

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22

? "t

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4 13

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299 398 423 448 473 498 523 548 573 598 623

EMISSION ( n m )

FIG. 3 . Isometric projection and monochromatic image o f a hexane extract from a mixture of Flavobacterium species.

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R O S S I , MOSS, AND WARNER

470

45 1

432

4 13

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318

299 398 423 448 473 498 523 548 573 598 623

EMISSION ( n m )

FIG. 4 . ether extractfrom a mixture of Flavobacterium species.

Isometric project ion and monochromatic image of an

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4 7 0

45 1

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4 13

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3 18

299 398 423 448 473 498 523 548 573 598 623

EMISSION ( n m )

FIG. 5. Isometric projection and monochromatic image of a 1:l ether:hexane extract from a mixture of Flavobacterium species.

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These o b s e r v a t i o n s , of c o u r s e , assume t h a t s p e c t r a l s h i f t s and

d i f f e r e n c e s i n t h e quantum e f f i c i e n c y of f l u o r e s c e n c e of t h e

pigments a re small among the various solvents. On the bas i s of the

preceding observations, the ethanol and ether solvents seem t o be

t h e b e s t f o r e x t r a c t i o n of t h e f l u o r e s c e n t pigments from t h e s e

organisms. Since t h e type and r e l a t i v e amounts of pigments

extracted by these two solvents d i f f e r , some combination of e the r

and ethanol should ex t r ac t the most pigments.

Two d i f f e r e n t extract ion procedures which used both e the r and

e t h a n o l as e x t r a c t i o n s o l v e n t s were e v a l u a t e d . I n t h e f i r s t

method, two e x t r a c t i o n s w i t h 1 m l of e t h a n o l were fo l lowed by a

s i n g l e e x t r a c t i o n w i t h 2 m l of e t h e r . A f t e r e x t r a c t i o n w a s

completed, the ether and ethanol were combined and analyzed using

v ideo f luo romet ry . An i s o m e t r i c p r o j e c t i o n and a monochromatic

image of t h i s e x t r a c t a r e provided i n F igu re 6. When t h i s EEM i s

compared t o the EEMs from the ex t r ac t s obtained with pure ethanol

(F igu re 1) and pure e t h e r (F igu re 2 ) , i t can be observed t h a t t h e

EEM of Figure 6 more closely resembles the ethanol e x t r a c t than the

e t h e r e x t r a c t s i n c e ve ry l i t t l e of t h e b l u e s h i f t e d pigment was

recovered. Hence, a s ing le extract ion with e the r i s not s u f f i c i e n t

t o recover a l l the e the r extractable pigments.

The second procedure used three consecutive extract ions with a

s o l v e n t mix tu re c o n t a i n i n g bo th e t h e r and e t h a n o l . I s o m e t r i c

project ions and monochromatic images of the EEMs of the ex t r ac t s

c o l l e c t e d u s i n g t h i s procedure a r e shown i n F igu res 7 and 8. The

highest i n t e n s i t y of fluorescence was observed when a 1:l mixture

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470

45 1

432

4 13

394

375

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337

3 18

299

FIG. 6. Isometric projection and monochromatic image o f the EEM o f an e x t r a c t from a mixture o f Flavobacterium-species obtained by extracting twice with 1 m l o f ethanol and once with 2 m l o f e ther .

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1 4 7 2

m E C

z k

Y

2 a t, U x W

R O S S 1 MOSS AND WARNER

470

45 1

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4 13

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375

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337

3 18

299 398 423 4 4 8 473 498 523 548 573 598 623

EMISSION ( n m )

FIG. 7. Isometric projection and monochromatic image of the EEM o f a 7:3 ethano1:ether extract from a mixture of Flavobacterium species.

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n E C Y

%

L 'j:

c( c a

w


470

45 1

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4 13

394

37s

356

337

3 10

299 398 423 440 473 490 523 f 4 0 373 S90 623

EMISSION (nn)

FIG. 8. Isometric projection and monochromatic image of the EEM o f a 1 : l ethano1:ether e x t r a c t f rom a mixture o f Flavobacterium species.

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of e t h e r and e t h a n o l w a s used. T h i s s o l v e n t m i x t u r e produced a

successful e x t r a c t of the pigments which were present i n both the

e the r and ethanol ex t r ac t s alone.

I d e n t i f i c a t i o n of Flavobacterium and Sphingobacterium Based on

Extracted P i g m e

The f l u o r e s c e n c e s p e c t r a of t h e e x t r a c t s from each o f t h e

b a c t e r i a l i s t e d i n Table I are shown i n F i g u r e s 9 and 10. Note

t h a t t h e t h r e e e x t r a c t s i nc luded i n F igu re 9 have s p e c t r a l

c h a r a c t e r i s t i c s s i m i l a r t o each o t h e r i n t h a t t h e s e E E M s a r e

characterized by broad emission spec t r a with maxima between 450 nm

and 500 nm. The e x t r a c t EEMs shown i n Figure 10, however, tend t o

show s l i g h t l y more e x c i t a t i o n s t r u c t u r e , o r have o t h e r s p e c t r a l

f e a t u r e s which, upon c l o s e examina t ion , make them unique as

compared t o t h e E E M s shown i n F igu re 9. The e x t r a c t of 5

meningosepticum appears a t f i r s t t o be somewhat s imi la r t o the EEMs

of F igu re 9 . However, i t has been grouped s e p a r a t e l y due t o a

s l i g h t s h i f t i n the fluorescence emission maximum toward the low

wavelength p o r t i o n o f t h e EEM. This n a t u r a l grouping of t h e

ex t r ac t fluorescence spec t r a is p a r t i c u l a r l y i n t e r e s t i n g when one

c o n s i d e r s t h a t t h e t h r e e organisms from which t h e e x t r a c t s of

Figure 9 were obtained ( i .e . , & thalpophilum, multivorum and S_

mizu tae ) have been found by o t h e r workers t o be s i m i l a r on t h e

b a s i s o f sph ingophospho l ip id and menaquinone c o n t e n t

However, spiri tovorum, which has a l s o been found t o be r e l a t e d

t o S_ mizutae i n terms of i ts chemical composition, d id not appear

t o have a v e r y s i m i l a r p r o f i l e o f f l u o r e s c e n t pigments when

3 4 4

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COMPARISON OF FLUORESCENT PIGMENTS

a

1475

T 79

40 w +

2 0 5

0 7 0

b T ' ' ' I

C

FIG. 9. Isometric projections of the E E M s of 1:1 ethano1:ether extracts from the first growth of: (a) thalpophilum; (b) 5 pltivorum, and; (c) S. mizutae.

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R O S S I , MOSS, AND WARNER 1 4 7 6

a

b

FIG. 10. Isometric project ions of 1:l ethano1:ether e x t r a c t s from t h e f i r s t growth o f : (a) meningosepticurn; (b) oderatum; ( c ) F- breve ; (d) 5 s p i r i t o v o r u m , and; (e) F- indologenes.

compared w i t h t h e o t h e r members of t h e Sphingobacter ium l i k e

organisms. Moreover, when compared t o each other , each of the EEMs

shown i n F i g u r e 1 0 a p p e a r t o b e u n i q u e . T h e r e f o r e , i t i s

r easonab le t o s p e c u l a t e t h a t p r o v i d i n g t h e e x t r a c t i o n procedure

y i e lds reproducible r e s u l t s , the EEMs of the ex t r ac t s may be useful

f o r the i d e n t i f i c a t i o n of the organisms tes ted. As a tes t of t h i s

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1477 COMPARISON OF FLUORESCENT PIGMENTS

C

398

d

39

e

F i g u r e 10 ( c o n t i n u e d )

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h y p o t h e s i s , e x t r a c t s were o b t a i n e d from new growths of t h e s e

o rgan i sms , a s d e s c r i b e d i n t h e p r o c e d u r a l s e c t i o n of t h i s

manusc r ip t , and F o u r i e r t r a n s f o r m based p a t t e r n r e c o g n i t i o n w a s

a p p l i e d d i r e c t l y t o t h e E E M s of t h e e x t r a c t s i n an a t t e m p t t o

ident i fy each of the species tes ted.

The r e p l i c a t e ex t r ac t s of the organisms a re shown i n Figures

11 and 1 2 . Once a g a i n , t h e s e E E M s have been grouped i n t o two

categories , those being the Sphingobacterium l i k e e x t r a c t s and the

remaining ex t r ac t s . By comparison of the e x t r a c t s shown i n Figures

11 and 1 2 t o t h o s e shown i n F i g u r e s 9 and 1 0 , i t can be s e e n t h a t ,

i n g e n e r a l , t h e r e s u l t s were r e p r o d u c i b l e f o r t h e i n d i v i d u a l

organisms. One n o t a b l e e x c e p t i o n t o t h i s o b s e r v a t i o n i s F-

meningosepticum, t h e e x t r a c t of which c o n t a i n s a much h i g h e r

r e l a t i v e concentration of a pigment with an emission maximum near

400 nm t h a n t h e f i r s t e x t r a c t of t h i s organism was observed t o

c o n t a i n . Fu r the rmore , S. mizu tae is s l i g h t l y d i f f e r e n t t h a n i t

appeared t o be i n t h e f i r s t e x t r a c t i o n i n t h a t a pigment which

e x c i t e s i n t h e 450 nm r e g i o n i s now c l e a r l y v i s i b l e . I n s p i t e o f

t h i s s l i g h t i r r ep roduc ib i l i t y , the S. mizutae e x t r a c t s t i l l appears

(by v i s u a l e v a l u a t i o n ) t o be more s i m i l a r t o t h e F. t ha lpoph i lum

and multivorum ex t r ac t s than it i s t o the remaining ex t r ac t s .

I n addi t ion t o evaluating the po ten t i a l f o r i d e n t i f i c a t i o n of

Flavobacterium species by a v i s u a l inspection of the e x t r a c t EEMs,

a F o u r i e r t r a n s f o r m based p a t t e r n recogni t ion algorithm w a s

applied t o the data. I n t h i s p a r t of the study, the e x t r a c t s from

the second growths were considered as unknowns, and the f i r s t s e t

12

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COMPARISON OF FLUORESCENT PIGMENTS

a

1479

T 43

b 513

i 4 4 L

C

FIG. 11. Isometric projections of the E E M s of 1:l ethano1:ether extracts from the second growth of: (a) thalpophilum; (b) multivorum, and; (c) & mizutae.

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ROSSI, MOSS, AND WARNER 1480

a 76

5 7 ,

38 $ I- l-4

b

FIG. 12. Isometric projections of 1:l ethano1:ether extracts from the second growth of: (a) F. meningosepticum; (b) F. oderaturn; (c) F. breve; (d) F. spiritovorum, and; (e) 5 indologenes.

of extracts (i.e., those shown in Figures 9 and 10) were used as a

library of standards. The pattern recognition results obtained

when the unknowns were compared to the standards are summarized in

Table 111. Note that with the exception of 5 meningosepcicug and

S. rnizutae, each of the unknowns was correctly identified. The

failures resulted from slight variations in the fluorescence

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C

d 160

t T 120

e

W I-

7 E

39

Figure 12 (continued)

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1482 ROSSI, EIOSS, AND WARNER

TABLE 3

Summary of Pattern Recognition Results

Unknown Bacterium Suggested Identification


Flavobacterium indologenes

Flavobacterium meningosepticum


Flavobacterium odaratum



SphinRobacterium mizutae

Flavobac ter ium breve

Flavobacterium indologenes



Flavobacterium odaratum




patterns of the replicate extracts. meningosepticum w a s

incorrectly identified as multivorum, and mizutae was

incorrectly identified as F. breve with F. thalpophilum and S_

mizutae being returned by the algorithm as somewhat less likely

possible identities. In the cases of these incorrect

identifications, it was clear from the output of the pattern

recognition algorithm that the suggested identifications were

not to be viewed with the same confidence level as the suggested

identifications in the cases where the identifications proved to be

correct. Therefore, there is very little chance that an incorrect

identification would be made based on these data.

Since the pattern recognition library consisted of only eight

members, each of which was a correct identification for one of the

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unknowns, the significance of the 75% success ratio observed in the

pattern recognition study must be questioned. It is useful,

therefore, to calculate the probability that six of the eight

unknowns would have been correctly identified by a random matching

with the standards. Such a calculation reveals that there is only

an 8.19 X chance that this success ratio would have been

observed for a random matching13 . Hence, it is apparent that

although the collection of standards with which the unknowns were

compared, the 75% success ratio observed is a reliable indicator of

the utility of this approach to bacterial identification.

CONCLUSIONS

The extraction scheme developed in this study was shown to be

useful for the isolation of fluorescent pigments from

Flavobacteriug and Sphingobacterium species. The identities of the

extracted pigments were not determined in the present study. The

complexity of the fluorescence spectra and the observation that no

single solvent was capable of extracting all of the observable

pigments leads to the conclusion that a mixture of compounds with

different chemical properties are being extracted. Two main

categories of pigments, carotenoids and flexirubins, have been

previously reported to exist in the Flavobacterium genus

However, the data presented in this study are insufficient for any

attempt at identifying the pigments.

3,14,15

At least two conclusions can be drawn from the data presented.

First, the fluorescence characteristics of the species 41,

multivorum, F. thalpophilum and S. mizutae are in agreement with

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independent studies which have shown other types of chemical

relatedness between these organisms. However, fluorescence

characteristics of S . mizutae and spiritovorum extracts were

found to be quite different in spite of the fact that F- spiritovorum has been observed to resemble mizutae in other

aspects of its chemical composition . 4

The second conclusion is that video fluorometry coupled with

advanced data reduction techniques such as the pattern recognition

algorithm used in this study, is a useful tool for the automatic

identification of those microorganisms with extractable fluorescent

pigments. In the present study, 75% of the organisms tested were

correctly identified. No attempt was made to examine bacterial

cell suspensions directly for fluorescence since scattered light

interferences would prohibit the use of the VF for recording the

fluorescence spectra of such turbid samples. In addition to

instrumental limitations of the VF, the extremely high turbidity of

whole cell suspensions used for extraction would produce a severe

inner filter effect, thereby making direct examination of the

cellular fluorescence impractical using most types of fluorometers.

ACKNOWLEDGEMENTS

The study reported in this manuscript was supported by a grant

from the National Institutes of Health (AIL9916). Isiah M. Warner

is also grateful for support from an NSF Presidential Young

Investigator Award (CHE-831675).

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1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

REFERENCES

Holmes, B., Owen, R. J., and McMeekin, T. A., Flavobacterium. In Bergey's Manual of Systematic Bacteriology, N. R. Krieg, J. G. Holt, Eds., Williams & Wilkens, MD, 1984, pp 353-361.

Reichenbach, R., Kohl, W., Bottger-Vetter, A., and Achenbach, H., Flexirubin-Type Pigments in Flavobacterigm. Arch. Microbiol., 126, 291-293 (1980).

Yabuuchi, E., Kaneko, T., Yano, I., Moss, C. W., and Miyoshi, N., Sphinpobac teriug gen. nov., Sphingobacterium spiritovorum comb. nov., Sphinpobacterium multivorum comb. nov., Sphingobacterium mizutae s p . nov. and Flavobacterium indologenes sp. nov.: Glucose-nonfermenting gram-negative rods in CDC groups IIK-2 and IIb. Int. J. Sklyst. Bacteriol., 2, - --_ -___--_--_ -____-_

580-598 (1983).

Dees, S. B., Carlone, G. M., Hollis, D., and Moss, C. W., Chemical and Phenotypic Characteristics of Flavobacterium ---- thalpphilum compared with other Flavobacterium and - Sphingobacterium -- species. Int. J. Syst. Bacteriol, 2, 16-22 (1985).

Johnson. D. W.. Gladden. J. A. , Callis. J. B.. and Christian, G. D., Video Fluorometer. Re;. Sci. instrum., so, 118-126 (1979).

Warner, I. M., Fogarty, M. P., and Shelly, D. C., Design Considerations for a Two-Dimensional Rapid Scanning Fluorimeter. Anal. Chim. Acta, 109, 361-372 (1979). Shelly, D. C., Quarles, J. M., and Warner, I. M., Identification of Fluorescent Pseudomonas species. Clin. Chem., 26, 1127-1132 (1980).

Shelly, D. C., Warner, I. M., and Quarles, J. M., Multiparameter Approach to the Fingerprinting of Fluorescent Pseudomonads. Clin. Chem., 26, 1419-1424 (1980).

Shelly, D. C., Quarles, J. M., and Warner, I. M., Preliminary Evaluation of Mixed Dyes for Fingerprinting Non-Fluorescent Bacteria. Anal. Lett., 14 (B13), 1111-1124 (1981). Shelly, D.C., Warner, I. M., and Quarles, J. M., Characterization of Bacteria by Mixed-Dye Fluorometry. Clin. Chem., 29, 290-296 (1983).

Rossi, T. M., and Warner, I. M., Bacterial Identification Using Fluorescence Spectroscopy. Instrumental Methods for Rapid gicrobiological Analysis, VCH Publishers, FL, 1985, pp 1-50.

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1486 ROSSI , MOSS, AND WARNER

1 2 . Rossi, T. M . , and Warner, I. M. , Pattern Recognition of Two- Dimensional Fluorescence Data Using Cross Correlation Analysis. Accepted for publication in Applied Spectrosc..

13. Bevington, P. R., Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill Book Company, NY, 1969.

14. Meckel, R. A., and Kester, A. S., Extractability of Carotenoid Pigments from Non-Photosynthetic Bacteria with Solvents and Detergents: Implications for the Location and Binding of the Pigments. J. Gen. Microbiol., 120, 111-116 (1980).

15. Achenbach. H., Bottger-Vetter, A., Fautz, E., and Reichenbach, H., On the Origin of the Branched Alkyl Substituents on Ring B of Flexirubin-Type Pigments. Arch. Microbiol., 3.32, 241- 2 4 4 (1982).

Received May 15, 1986 Accepted June 16, 1986

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species

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