The isolation of sucrose-fermenting Salmonella mbandaka

Veterinary Microbiology, 37 ( 1993 ) 181-185 Elsevier Science Publishers B.V., Amsterdam

181

Short Communication

The isolation of sucrose-fermenting Salmonella mbandaka R.L. Reid, R.C. Porter and H.J. Ball Department of Agriculturefor Northern Ireland, VeterinarySciences Division, Stormont, Stoney Road, Belfast, UK (Accepted 29 December 1993) ABSTRACT During media trials to evaluate the use of Brilliant Green Agar for the primary recognition of Salmonella, strains presenting fermentation reactions were observed. All fermenting strains (84 out of 145 ) belonged to the serotype Salmonella mbandaka (58%), and the activity was expressed on three batches of Brilliant Green Agar and one of Xylose-Lysine Desoxycholate Agar. It was established using individual lactose and sucrose broth that the reaction in these media was due to sucrose fermentation. The most frequently isolated Salmonella in this laboratory during 1990 was S. mbandaka (61%) i.e. 65 of the 106 isolates during this period. Primary differentiation of Salmonella from other members of the family Enterobacteriaceae on media incorporating sucrose would have resulted in 36% of Salmonella isolates not being recognised. BGA and XLD agar therefore would not be suitable for primary isolation of Salmonella from clinical material with such a high percentage of the major isolate, S. mbandaka, having the ability to ferment surcrose.

INTRODUCTION

Studies have shown that some serotypes of Salmonella have the ability to ferment sucrose, although Wohlhieker et al. ( 1975 ) considered it to be a rare occurrence. Johnston et al. (1976) demonstrated the ability ofS. tennesse not only to ferment sucrose but also lactose, and Ceddia et al. (1987) recorded sucrose fermentation in S. arizona. The original isolate ofS. mbandaka was made in the Belgian Congo in 1948 (Kauffman and Reul, 1949). From 1948 to 1985, 3497 isolates of this serotype were cultured from a wide variety of sources worldwide (Fantasia et al. 1989). In Italy there was an increase from one isolate in 1979 to 118 in 1986 (67% of human origin), and in this laboratory the number of isolates from animal (mainly poultry) sources has increased annually from two in 1982 to 68 in 1991 (unpublished results). Correspondence to: R.L. Reid, Department of Agriculture for Northern Ireland, Veterinary Sciences Division, Stormont, Stoney Road, Belfast BT4 3SD, UK. 0378-1135/93/$06.00 © 1993 Elsevier Science Publishers B.V. All fights reserved.

182

R.L. REIDETAL.

During a comparison of Brilliant Green Agar (BGA) with Desoxycholate Citrate Agar (DCA) as a differential medium for preliminary identification of Salmonella spp., a high percentage of fermenting S. mbandaka strains were observed. The present study was undertaken to identify the source of fermentation and to screen Salmonella isolates for the ability to ferment this source. MATERIAL A N D METHODS

Bacterial cultures All S. mbandaka cultures originated from animal specimens processed by the laboratory during the period 1988 to 1991, and had been stored at - 70 °C on Protect (Technical Services Ltd, Bury). These cultures were isolated from a wide variety of specimens, i.e. viscera, faeces, dust and box-liners. The majority of which came from 42 different poultry flocks (including 36 chicken and 6 turkey) located throughout Northern Ireland. The other Salmonella TABLE1 The presumptive differentiation of fermenting and non-fermenting strains of Salmonella on Brilliant Green Agar Salmonella serovar

Non-fermenting strains (red colonies )

Fermenting strains (yellow colonies )

mbandaka agona alachan albany berta

10 6 1 2 1 4 1 1 3 15 3 1 1 1 2 1 1 1 1 1 1 2 18 1

15 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

bredeney cape

choleraesuis derby dublin enteridits give goldcoast heidelberg infantis lexington

meleagridis muenster newport ohio roterberg senfienburg

typhimurium zongo

ISOLATIONOF SURCROSE-FERMENTINGSALMONELLA MBANDAKA

18 3

serovars utilised for this study were representative of the range of Salmonellae processed by this laboratory over a similar period. The cultures were isolated mainly from clinical material of animal origin, some from environmental samples and a few from samples of meal. The cultures were inoculated onto blood agar and checked for purity prior to examination on the test media. The Salmonella serotypes were identified with commercial Salmonella agglutinating sera. (Wellcome Diagnostic Ltd, Dartford, UK; and Pro-Lab Diagnostic, Bury, Lancashire, GB ). AP 120E (Bio-Merieux, Miareg-L'Etoile, France) kits were used to confirm the initial serological identification of the sucrose-fermenting strains in Table 1. Of the 145 S. mbandaka cultures examined 140 were isolated from poultry, three isolates were from specimens taken at the local zoo (egrit, spoonbill and pheasant), one was an ovine isolate and one isolate was from a meal sample. Reference cultures from the National Collection of Type Cultures (NCTC, Colindale, London) of S. typhimurium 0074, E. coli 9001, S. mbandaka 7892 and Klebsiella pneumoniae 9633 were also included as positive and negative controls.

Media The solid agar media examined were: two batches of BGA supplied by Oxoid Ltd, Basingstoke; one batch of BGA supplied by Difco Ltd, Detroit, Michigan; two batches of DCA supplied by Oxoid Ltd, one batch of DCA supplied by Difco Ltd and one batch of Xylose-Lysine Desoxycholate Agar (XLD) supplied by Oxoid Ltd. Batches of Peptone water broth (Oxoid Ltd) had 1% of lactose or sucrose (British Drug Houses, Poole, Dorset) added. All media were prepared according to the supplier's instructions. Each agar medium was inoculated by streaking with a standard 10/tl disposable sterile loop from the Blood Agar culture used to establish purity. Each isolate was inoculated into both lactose and sucrose broths to demonstrate which carbohydrate was being utilised in the selective media. All media were incubated at 37°C for 18 hours. RESULTS

During preliminary studies yellow colonies, indicating lactose or sucrose fermentation, were observed in 15 out of 25 S. mbandaka strains when grown on the BGA (Table 1 ). The initial sucrose-fermenting S. mbandaka strains were identified as Salmonella species with the AP 120E kit. Further investigations using seven different batches of three agar media confirmed that 84 out of 145 S. mbandaka isolates fermented lactose and/or sucrose (Table 2). Results were consistent between the three batches of BGA and between three batches of DCA. No other Salmonella serotypes examined exhibited fermentation activity. Cultures of the isolates in lactose and sucrose broths demonstrated that the fermentation was confined to sucrose and not lactose.

184

~.L. REID ET AL.

TABLE 2 C o m p a r i s o n o f three differential m e d i a u s e d for the p r e s u m p t i v e diagnosis Number of strains

of Salmonella

MEDIA DCA (three b a t c h e s )

BGA (three b a t c h e s )

XLD ( 1 batch)

-

-

-

-

-

-

S. mbandaka

N T C C 7892

1

S. mbandaka

61

S. mbandaka

84

-

+

+

S. agona

1

-

-

-

S. bredeney

1

-

-

-

S. choleraesuis

1

-

-

-

S. dublin

2

-

-

-

S. enteriditis

6

-

-

-

S. give

1

-

-

-

S. goldcoast

1

-

-

-

S. heidelberg

1

-

-

-

S. indiana

1

-

-

-

S. infantis

1

-

-

-

S. kentucky

1

-

-

-

S. livingstone

1

-

-

-

S. montevideo

1

-

-

-

S. ohio

1

-

-

-

S. oranienburg

1

-

-

-

S. panama

1

-

-

-

S. taksony

1

-

-

-

S. thompson

1

-

-

-

S. typhimurium

6

-

-

-

1

-

-

-

1

+

+

+

1

+

+

+

S. typhimurium

N T C C 0074 E. coli

N T C C 9001 K. pneumoniae

N T C C 9633

+ cultures d e m o n s t r a t i n g f e r m e n t a t i o n ie suspect n o n - S a l m o n e l l a - cultures d e m o n s t r a t i n g non-ferm e n t a t i o n ie suspect S a l m o n e l l a . D C A = Desoxycholate Citrate Agar. B G A = Brilliant G r e e n Agar. X L D = Xylose-Lysine Desoxycholate Agar.

DISCUSSION

There are several biochemical reactions common to the majority of Salmonella species which are utilised in the presumptive identification to differentiate this genus from others of the family Enterobacteriaceae. In this study DCA, which incorporates lactose, and BGA and XLD, which incorporate lactose and sucrose, were used. Some Salmonella strains have been noted to ferment sucrose (Wohlhieker et al., 1975, Johnston et al., 1976, Ceddia et al.,

ISOLATIONOFSURCROSE-FERMENTINGSALMONELLAMBANDAKA

18 5

1987, F. Kauffmann, 1949, Lennette, 1985). This study demonstrated that sucrose in BGA and XLD was being fermented by 58% of S. mbandaka isolates examined. The fermentation of the sucrose was masking the inability of strains to ferment lactose, which is the key differential characteristic on these two media. As S. mbandaka was the major Salmonella serotype isolated (61% of isolates) in this laboratory in 1990 and 199 l, sole utilisation of BGA and/ or XLD in the culture procedure would have significantly reduced the number of Salmonella isolated. Since the isolation of sucrose-fermenting Salmonella appears to be a rare occurrence and the source of such strains in the present study was limited to a single serotype isolated largely from avian sources, it is probable that these strains originated from a single source. The wide geographical distribution throughout Northern Ireland of the fermenting S. mbandaka strains within the poultry industry also indicated poultry feed to be the probable source.

REFERENCES Ceddia, T., Gioia, G., Mancini, A., Natellis, C., and Oliva, B., 1987. Simplified identification of bacteria belonging to the genus Salmonella. Zbl. Bakt. Hyg. A 266: 438-442. Fantasia, M., Pontello, M., Filetici, E. and Aureli, P. 1989. Salmonella mbandaka isolated in Italy 1979-1986. Microbiologica, 12: 49-54. Johnston, E.M., Wohlhieter, J.A., Placek, B.P., Sleet, R.B. and Baron, L.S., 1976. Plasmid-determined ability of a Salmonella tennessee strain to ferment lactose and sucrose. J. Bacteriol., 125: 385-386. Kauffman, F., 1969. The Bacteriology of Enterobacteriaceae. Munksgaard-Copenhagen, Scandinavian University Books p 66. Kauffman, F. and Reul, R. 1949. Two new Salmonella types from the Belgian Congo. Acta Path Micro (Scand) Vol. XXVI, 2, 335-336. Lennette, E.H., Balows, A., Hauster, Jr. W.J., Shadomy, J.H. 1985. Manual of Clinical Microbiology 4th Ed. Am. Soc. Microbiol. Washington, D.C. p 266. Wohlhieter, J.A., Lazere, J.R., Snellings, N.J., Johnson, E.M., Synenki, R.M. and Baron, L.S. 1975. Characterization of transmissible genetic elements from sucrose-fermenting Salmonella strains. J. Bacteriol., 122: 401-406.