Identification and flagellation of coryneform bacteria from poultry litter1

J. ANTHEUNISSE A N D H. VONKEMAN~ Laborarory of Microbiology, Agricultural Un i~~ers i iy , Wageningen, The Netherlands

Accepted July 21, 1975

ANTHEUNISSE, J . , and H. VONKEMAN. 1975. Identification and flagellation of coryneform bac- teria from poultry litter. Can. J. Microbiol. 21: 1798-1802.

Thirty coryneform isolates from poultry litter were identified and checked for motility and flagellation. Twenty-seven formed a yellow pigment and 3 were orange. Twenty-three yellow strains were found to be Arrhrobacrer ciireus, although starch was hydrolyzed by these strains. Four strains, including the three mainly pale yellow isolates, grew on citrate plus an ammonium salt and were classified as A . aurescens. Three orange strains were found to be Brevibacierirrm linens. Only six strains ofA. ciirerrs were motile. These strains displayed flagellated rods after 1 day and flagellated cocci after 5 days incubation. The flagellar shape and arrangement were studied. Non-motile strains never showed flagella after staining.

ANTHEUNISSE, J . , et H. VONKEMAN. 1975. Identification and flagellation of coryneform bacteria from poultry litter. Can. J . Microbiol. 21: 1798-1802.

Nous avons identifik et verifiC la mobilitC et la flagellation chez 30 isolats coryneformes de la litiere d'un poulailler. Vingt-sept forment un pigment jaune et 3 sont orange. Vingt-trois isolats jaunes sont classes comme Arillrobacfer ciireus, bien que I'amidon est hydrolise par c e s isolats. Quatre isolats, incluant les trois ayant un pigment jaune pile, se dkveloppent sur le citrate additionnt de sel d'ammonium et sont classifiCs comme Ctant A. alrrescens. Trois isolats orange appartiennent au groupe Brevibacierirrm linens. Seulement six isolats de A . ciirerrs sont mobiles. Ces isolats montrent des bitonnets flagellks apres 1 journee d'incubation et des cocoTdes flagellks apres 5 jours d'incubation. Les formes flagellkes et I'arrangement furent etudiks. Les isolats non mobiles ne montrent jamais de flagellation apr t s coloration.

[Traduit par le journal]

Introduction A survey of coryneform bacteria isolated from

various sources including soil, activated sludge, and cheese surfaces was made by Mulder et al. (1966). Three distinct groups of coryneforms were distinguished, viz. those belonging to the genus Arthrobacter, the orange bacteria of the Brevibacterium linens type isolated from cheese and fish surfaces, and a group of non-orange cheese coryneforms. The validity of this classi- fication was confirmed in studies on deoxyribo- nucleic acid analysis and in hybridization ex- periments (Crombach 1972, 1974~). The de- composition of uric acid and urea by these groups of coryneforms was tested by Antheu- nisse (1972). Schefferle (1965) described the iso- lation of 248 coryneform bacteria from built-up poultry litter. These microorganisms were pre- dominant among the total number of isolates and many of them formed yellow or orange pigments. Uric acid was decomposed by 33% of

'Received February 28, 1975. 'Present address: Unilever Research Laboratory,

Olivier v. Noortlaan 120, Vlaardingen, The Netherlands.

the isolated coryneforms. Most of these strains converted the uric acid only into urea. Organ- isms decomposing urea but not uric acid were also reported. Motile coryneform bacteria iso- lated from poultry litter were already observed by Schefferle in 1957. In the present paper the flagellar shape and arrangement of such isolates are described and compared with those of Ar- throbacter strains derived from soil (Antheunisse 1974).

Materials and Methods A compound sample of litter was taken from a poultry

run, a t environmental temperature of about 5 OC, a n d homogenized. Aliquots were diluted with sterile t ap water and disseminated on agar count plates. From the

and lo-' dilutions well-isolated colonies were randomly taken. A total of 139 colonies were subcultured on slants till microscopically pure: 133 (95.773 of the isolates were pigmented (cream, yellow, or orange). Thirty randomly selected strains of these isolates were further investigated.

The strains numbered 825-839 were obtained f rom agar plates containing meat extracts, 25 g; yeast extract, 0.1 g ; glucose, 0.2 g ; and soil extract, 1000 ml. Numbers 840-845 were obtained from the same medium with 4% sodium chloride added.

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ANTHEUNISSE AND VONKEMAN: CORYNEFORM BACTERIA FROM POULTRY LITTER 1799

Growth on citrate and ammonium chloride was tested in a liquid medium in tubes containing sodium citrate (5.5 HzO), 5 g; ammonium chloride, 1 g; K2HP04, 1 g; yeast extract, 50 mg; soil extract, 100 ml; and tap water, 900 ml; final pH 6.8. A mixture of nine vitamins was added after sterilization. After 7 days incubation a t 30 "C in a shaker, the turbidity was compared with that in the same medium without ammonium chloride.

The decomposition of uric acid and urea was tested according to Antheunisse (1972). The growth at high salt concentrations (8% and 15%) was ascertained in a liquid medium in tubes containing per litre: yeast extract, 3 g; glucose, 6 g; soil extract, 50 ml; sodium chloride, 80 g or 150 g, made up with tap water; final pH 7.0. After 6 days incubation in a shaker at 30°C the growth was compared with that in the medium without sodium chloride. Pigmentation was ascertained after 7 days in- cubation in the dark and in the light on an agar medium containing yeast extract, 0.7%, and glucose, 1%. The yellow strains were identified according to the key to the species of the genus Arthrobacter in Bergey's Manual (Breed er al. 1957).

Motility was checked with young cultures on agar slopes containing yeast extract, 0.7%, and glucose, 1%. The rods were incubated for 18-20 h at 28 "C and ob- served immediately after preparation of the suspension by a phase-contrast microscope. The flagella of rods and cocci of the motile strains were strained according to Gray (1926). Cleaning and pretreatment of slides were carried out as described by Antheunisse (1974).

Results Most of the isolates from poultry litter were

coryneform bacteria. Of the 30 representative coryneform strains investigated, 27 formed a yellow and 3 an orange pigment when growing in the light. In the dark, seven yellow and one orange strain were hardly pigmented. Most strains failed to grow in the citrate - ammonium salt medium (Table 1). Only four mainly pale yellow strains utilized these compounds for growth.

Nearly all of the strains showed rods with "snapping" division after 24 h incubation a t 28 "C on yeast extract (0.7%) and glucose (1.0%) agar, coccoid cells, after 5 days on soil extract agar with 0.1% yeast extract at 28 "C. They were Gram-positive and decomposed gelatin, starch, and uric acid. Only five, mainly pale yellow strains, possessed the enzyme urease. Nitrite was formed from nitrate by 24 strains. Nearly all of the strains tested were able to grow a t 10 "C but none of them grew a t 3(7 "C. Eleven strains grew abundantly in the presence of 8% sodium chloride, but only the 3 orange strains grew well with 15% sodium chloride in the medium. The

nutritional requirement, positive Gram stain, and ability to grow with 15% (after exposure to 18%) sodium chloride in the medium. Twenty- three of the yellow strains were identified as Arthrobacter citreus, with the only difference that all of the strains tested hydrolyzed starch. Three pale yellow strains and one yellow strain were classified as Arthrobacter aurescens.

Six strains of A. citreus were motile. The rods as well as the cocci of these strains were flagel- lated (Fig. 1-1 5). Young cultures of each strain showed a mixture of subpolar and "degenerated" peritrichous (Conn and Wolfe 1938) flagellar arrangements. The peritrichous rods possessed from one t o five flagella. The subpolarly flagel- lated rods were mainly monotrichous. Often two and sometimes three subpolar flagella were ob- served. Most of the flagella on rods and cocci had a wavelength of 1.7-1.9 pm. Many wave- lengths were found between 1.3 and 1.7 pm. The flagellation of these yellow strains is situated on the border line of the curly and normal flagellar type (Antheunisse 1974). Among the predom- inant flagellar type, rods and cocci with coiled, semicoiled, and, less commonly, undulant flagella (Leifson 1960) were observed. Many times a coil was observed a t the end of a flagellum. In spite of the presence of flagella, coccoid cells were rarely motile.

Discussion In contrast to the observations of Schefferle

(1965), that only one-third of the coryneforms from poultry litter decomposed uric acid, all the coryneforms tested in the present study conver- ted this compound. A small part (17%) of these strains showed urease acitivity, which agreed with Schefferle's results. Antheunisse (1972) reported the decomposition of uric acid by most of the coryneform bactiria from soil and sewage and by a high percentage of the orange isolates from cheese and sea fish. Most of the strains from soil and sewage decomposed urea, but the orange isolates (B. linens) showed hardly any urease activity.

Schefferle (1957) found a high salt tolerance of B. linens and of nearly all of the other coryne- form bacteria from poultry litter, which were mostly yellow-pigmented. According to that author, the high salt tolerance is of no use for

three orange strains were identified as Brevibac- terium linens because of their pigmentation, high

differentiating the two types of organisms. The present investigation shows that 19 out of 27

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TABLE 1

Characteristics of chromogenic coryneforms from poultry litter

Pigmentation in the: Growth on Nitrite Growth Decom- Strain citrate and Hydrolysis from Growth at with position No. Species light dark NH, salts of starch nitrate 10 "C 8% NaCl of urea Motility

826 A. citreus Yellow Cream - + + + - - - 829 " Yellow - + + + k - - 830 " Pale + + + - + - - -

yellow 832 " Cream - + + + - + - - 833 " Pale - + - + - - -

yellow 834 " Cream - + + + * - - 835 " Yellow - + + + - + - - 837 " Pale - + + + k - + 2.

0

yellow Z 838 " Pale - + + + - + - - ?

yellow Z 839 " Pale - + - + - - + 5

yellow n

840 " Yellow - + - + - - + s 841 " Cream - + + + k - - 5 8 43 " Yellow - + + + + + - r 844 - - - + - <

" Yellow + + o 845 " Cream - + + + - + - - r 846 " Yellow - + + + + - - N 847 " Cream - + + + - + - - - 848 - - - w

" Yellow + + + + VI 4

849 " Yellow - + - + - + - - 850 " Yellow - + - + + - + 851 " Cream - + + + * - - 853 " Yellow - + + + + - - 854 " Yellow - + - + - - + 827 A. aurescens Pale Cream + + + + + + -

yellow 828 Pale Cream + - + + + + -

yellow 836 Pale Cream + + + + - + -

yellow 842 Yellow White + + + + + - - 825 B. linens Orange Orange - - + + + - - 831 Orange Cream - - + - + + - 852 Orange Orange - - + + + - -

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ANTHEUNISSE AND VONKEMAN: CORYNEFORM BACTERIA FROM POULTRY LITTER 1801

FIGS. 1-15. Flagellar arrangement and shape of rods and cocci of Arthrobacter strains. Figs. 1-3, strain 839; Figs. 4-6, strain 840; Figs. 7-9, strain 844; Figs. 10-12, strain 850; Figs. 13-15, strain 854. Figs. 1 and 4. Rods with "degenerated" peritrichous flagella. Figs. 7 and 10. Rods with subpolar flagella. Fig. 2. Flagel- lum with small wavelengths (between 1.3 and 1.7 pm) on a coccus. Fig. 6. Coccus with a semicoiled flagellum and a flagellum with small wavelengths. Fig. 8. A coil at the end of the flagellum. Fig. 14. Coccus with a coiled flagellum and a flagellum with small wavelengths. Magnification 2500 x .

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CAN. J. MICROBIOL. VOL. 21, 1975

yellow Arthrobacter strains isolated from poultry litter grew poorly or not a t all in a medium supplied with 8% sodium chloride. These 19 strains resemble the soil arthrobacters which in earlier studies were found to have a low resis- tance to salt (Mulder et al. 1966; Crombach 1974b).

Motility of some of the coryneform bacteria isolated from poultry litter was already observed by Schefferle in 1957. The mixed, subpolar and "degenerated" peritrichous flagellation of the six motile yellow strains of A. citreus, as described in the present paper, is similar to that of the motile soil arthrobacters (Antheunisse 1974). The six motile strains show flagella with rather small wavelengths comparable with those of some yellow soil arthrobacters. Coccoid cells of arthrobacters isolated from soil and poultry litter are frequently flagellated. However, in both cases motility of cocci is rarely observed.

The initial concept that cocci of the genus Arthrobacter occurring in exhausted media are cyst-like end forms seems doubtful because of their growth in a logarithmic phase (Ensign and Wolfe 1964; Luscombe and Gray 1971 ; Crom- bach 1974b), the frequent presence of flagellated cocci (Antheunisse 1974), and the observation of Boylen (1973) that coccoid cells of A. crystal- lopietes do not differ from the rods in resistance against desiccation.

In the 1974 edition of Bergey's Manual R. M. Keddie considered A. aurescens as a "subjective synonym of A. globijbrmis." This was because of similar minimal nutritional requirement, cell wall composition, range of organic substrates utilized as the sole C source, and in the guanine- cytosine (G/C) ratio. Also, R. M. Keddie and M. Rogosa place Brevibacterium as a genus incertae sedis and note that B. linens is "related to Arthrobacter, particularly A. globijbrmis."

Acknowledgment

Thanks are due to Mrs. E.v.d. Pol-v. Soest and Mrs. C. Moller-Mol for technical assistance.

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