Differentiation and identification of human faecal anaerobic bacteria producing β-galactosidase (a new methodology)

ELSEVIER

Journal of Microbiological Methods 27 (1996) 25-31

Journal ofMicrobiological Methods

Differentiation and identification of human faecal anaerobic bacteria producing P-galactosidase (a new methodology) C. Favie?,

C. Neutb, C. Mizona’*, A. CortotC, J.-F. Colombel”,

J. Mizon”

“Laboratoire de Biochimie, Faculth de Pharmacie, 3, rue du Professeur Laguesse, B.P. 83, Lille 59006, France bLuboratoire de Bacte’riologie, Faculte’ de Pharmacie, Lille, France ‘Service des Maladies de 1 ‘Appareil Digestif et de la Nutrition, HGpital Huriez, Centre Hospitalier Rigional et Universitaire, Lille, France

Received 5 February 1996; revised 12 May 1996; accepted 11 June 1996

Abstract Bacterial glycosidase activities, specially P-galactosidase (P-gal), are reduced in faeces from patients with Crohn’s disease (CD). In order to test the hypothesis that an alteration of the colonic flora might be responsible for decreased faecal P-gal activity in CD patients, we developed a new methodology designed to differentiate and enumerate anaerobic bacteria producing P-gal. We used an enriched Columbia agar medium modified by addition of 3-bromo-4-chloro-5indolyl-P-ogalactopyranoside (X-gal) and tested it on different faecal bacterial strains. The presence of blue diffuse zones with various sizes surrounding bacterial colonies clearly differentiated Bijidobacrerium, Ruminococcus, Bacteroides and one of five strains of Lactobacillus tested from other P-gal positive bacteria. When the X-gal medium was applied to the analysis of normal human faecal flora, 5’7%?29 (mean+S.D.) of the colonies growing on this medium were surrounded by blue haloes. Among them, all colonies, characterized by a large blue diffuse ring, were identified as bifidobacteria. Keywords:

Faecal flora; Anaerobic bacteria; P-galactosidase; X-gal; Bijdobacterium

1. Introduction The analysis of colonic microflora in patients with Crohn’s disease (CD) has been approached by studying its metabolic activity, i.e. quantification of the specific products of bacterial metabolism or determination of microbial enzymes in faecal extracts [ 1,2]. Among the latter, exoglycosidases were especially studied [1,3]. We have recently demonstrated that, in active CD, these glycosidases, particularly ,B-galactosidase (P-gal), were strongly re-

*Corresponding author. Tel.: +33 20964045; fax: +33 20959009.

duced in faeces [4,5]. Those data were recently confirmed by Malin et al. [6]. In order to test the hypothesis that an alteration of the colonic flora might be responsible for decreased faecal P-gal activity in CD patients, we developed a new method using 3-bromo4-chloro-5-indolyl-P-ogalactopyranoside (X-gal) as chromogenic substrate. Our method improves an earlier described procedure [7] because it directly differentiates onto plates the anaerobic strains producing P-gal. Furthermore, the strains releasing extracellular P-gal produce large blue haloes surrounding the bacterial colonies. This procedure was applied to the qualitative and quantitative analysis of faecal samples from healthy

0167-7012/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO1 67-70 12(96)00925-6

26

C. Favier et al. I Journal of Microbiological Methods 27 (1996) 25-31

subjects. The bacterial colonies surrounded by blue haloes were further identified by conventional culturing techniques.

2. Materials

and methods

2.1. Origin of bacterial

strains

Clostridium pe$ringens (DE), Eubacterium alactolyticum (DG2), Bijidobacterium bijidum (MCY), Lactobacillus sp. (LB4 and LB8), E. coli (AMl) Streptococcus sp. (CF), Bacteroides fragilis (AM2) were isolated and identified according to classical procedures [8]. The reference strains, listed in Table 1, were kindly supplied by Dr. F. Gavini (INRA, Villeneuve d’Ascq, France). All strains were subcultured in Wilkins-West broth [9]. 2.2. Faecal samples The faecal samples from 13 healthy volunteers, (11 women, two men), mean age 33 years (range 18-58), having received no antibiotics for at least 3 months, were analyzed within 1 h following collection. 2.3. Culture medium Commercial Columbia medium (BioMCrieux, France) was supplemented with glucose (5 g l-l), cysteine hydrochloride (0.3 g 1-l) and agar (5 g 1-i). X-Gal (Interchim, France) (10 mg in 0.5 ml of NJ/-dimethylformamide) was further added to 100 ml of the above medium.

2.5. Methods The X-gal medium was inoculated with 0.1 ml of tenfold dilutions, i.e. either up to lop6 for the cultures of strains or 10m9 for faeces. For stools, approximately 1 g, exactly weighted, was suspended in 9 ml. All the dilutions were carried out with l/4 Ringer solution supplemented with 0.3 g 1~ ’ cysteine hydrochloride. After inoculation, plates were quickly placed into the anaerobic chamber. Pure strains were incubated at 37°C during 3 days, and faecal samples for 7 days. After completion, the intensity and size of the blue zones produced were noted within 10 min by two independent persons, using a semiquantitative ranking: (0) for white colonies without P-gal activity; (-) for blue colonies without blue halo; (2 + ): blue diffusion zone outside the colonies; (+): diameter of the ring <2 mm; (+ +): diameter between 2 and 6 mm; (+ + +): diameter 26 mm. For each faecal sample, colonies were counted by choosing plates with l-100 colonies and the results were expressed as cfu g-i wet faeces. All colonies with blue zones surrounding them were enumerated and some of them were subcultured for identification, using classical biochemical and morphological characteristics [8]. The Bijidobacterium strains were identified to the genus level. Furthermore, bifidobacteria count was determined from faecal samples on the selective and elective propionic acid medium (MB) described by Beerens [lo]. The expressed counts only took into account colonies with typical bifidobacterial morphology after Gram staining.

3. Results 2.4. Incubation

equipment 3.1. Optimization

An anaerobic chamber (Forma Scientific, Ohio, USA N2 75%, H, lo%, CO, 15%) was used. Anaerobic jars with GasPak system (BioMerieux, France) or AnaeroGen (Oxoid, Unipath, Dardilly, France) or individual incubation systems Generbag Anaer (BioMerieux, France) were also tested for anaerobic culture. Methylene blue test strips (BioMerieux, France) were used as indicators of anaerobiosis.

of the procedure

The ability of the medium to differentiate P-gal producing bacteria was first evaluated by culturing three different strains. Eubacterium alactolyticum (DG2) does not ferment lactose; Clostridium perfringens (DE) produces intracellular p-gal; Bifidobacterium bzjidum (MCY) is a lactose fermenter with a high P-gal activity in culture supematants (data not shown). After growing on the X-gal medium, DG2

C. Favier et al. I Journal of Microbiological Methods 27 (1996) 25-31 Table 1 Comparative

responses

of bacterial

21

strains on X-gal medium

Strains

Code

0/-l+/++/+++

Bijidobacterium bifidum

CUETM 89127 CUETM 89/291 CUETM 89/278 CUETM 89/286 ATCC 15696 CUETM 89/220 CUETM 89/160 CUETM 90/103 T CUETM 89/ 157 CUETM 89/ 177 CUETM 89/216 CUETM 89/276 CUETM 89/11 T CUETM 89/19 T CUETM 89/14 T CUETM 89/12 T ATCC 27669 CUETM 89/96 T CUETM 90/ 136 CUETM 89/158 CUETM 89/20 T ATCC 27679 CUETM 89/141 CUETM 89/219 CUETM 89/16 T CUETM 89/29 CUETM 89/178 ATCC 29176 ATCC 29149 ATCC 25285 AM2 LB4 LB8 CIP 7139 CIP 7135 IPL 1627 ATCC 2636 CIP 54127 AM1 CIP 5855 CF ATCC49630

++ +++ +++ +++ +++ +++ +++ ++ + +

Bijdobacterium breve

Bijidobacterium longum or infantis

Bifdobacterium Bijdobacterium Bifdobacterium Bifidobacterium

longum infanris adolescentis angulatum

Bifidobacterium catenulatum

Bifidobacterium dentium

Bijidobacterium pseudocatenulatum

Ruminococcus lactaris Ruminococcus gnavus Bacteroides fragilis Lactobacillus sp. Lactobacillus plantarum Lactobacillus brevis Zactobacillus acidophilrrs Clostridium perfringens Escherichia coli Enterococcus faecalis Streptococcus sp. Peptostreptococcus hydJpogenalis

+ +++ ++ + ++ ++ + +++ + + +++ + +++ + ++ ++ ++ + + +

0, white colonies without p-gal activity; - , blue colonies without blue halo; 5 + , blue diffusion zone outside colonies ( + , diameter ring <2 mm; + + , diameter between 2 and 6 mm; + + +, diameter ~6 mm). CUETM, Collection Unite Ecotoxicologie Microbienne (France). ATCC, American Type Culture Collection. CIP, Collection Institute Pasteur (Paris, France). IPL, Institute Pasteur Lille (France). T, type strain.

C. Favier er al. / Journal of Microbiological Merhods 27 (1996) 25--11

28

colonies were white (0), DE colonies were blue (-), MCY colonies were blue with a large diffuse zone (+ +). When plated together, the three strains were easily distinguished by their own colour and the presence (or not) of a blue zone surrounding the colonies. Decreasing the substrate concentration in the medium to 50 mg 1-l led to the same result, but the staining was intensified by using 100 mg 1-l substrate. When less than 100 colonies per plate were present, the blue zones were more easily detected. With regard to the four different anaerobic incubation systems, our method was reliable when the plates were incubated either in an anaerobic chamber or with the Generbag system. In contrast, no colour could be observed with GasPak or AnaeroGen systems. 3.2. Release of P-gal by reference

strains

Faecal strains using (or not) lactose as a substrate for growth were inoculated on the X-Gal medium (Table 1). All the bifidobacterial species produced blue rings. The strains of Bijdobacterium b$idum and Bifidobacterium breve exhibited large blue haloes (+ +, + + +). A blue zone was observed for Ruminococcus. larger for R. lactaris than for R. gnavus. Bacteroides fragilis showed colonies with a Table 2 Counts of faecal bacteria Samples

expressed

as lo9 of number of bacteria

A

B

ColoniesOl-I+!++/+++

Colonies

per g wet weight of faecal material cultured on X-gal medium C (%? (38.9) (38.0) (100.0) ( 100.0)

(70.8)

3.55 1.29 0.85 3.09 3.63

0.79 7.24 2.34 0.33 3.47 20.89 0.56 2.63

(42.5) (46.7) (69.0) (41.8) (69.3) (91.2) (13.1) (43.7)

0.07 7.24 0.50 0.20 0.56 2.09 0.14 0.28

(8.9) (100.0) (21.4) (60.6) (16.1) (10.0) (25.0) (10.6)

4.525.6

(57k29)

1.8-f2.1

(48238)

6 7 8 9 10 11 12 13

1.86 15.49 3.39 0.79 5.01 22.91 4.27 6.02

MeankS.D.

7.727.6 to B X 100/A. to CX 100/B).

The faecal samples contained variable numbers of bacteria producing blue zones surrounding the colonies (13-100% of the total count) (Table 2). They represented, on the average, 57%t29 (meaniS.D.) of total colonies. Among them, 52%+38 formed blue colonies with a small diffusion zone (+ ). The 20 colonies that were identified, belonged to the genera Bacteroides (fragilis group, strict0 sensu) (n = 14) and Prevotella (n=6). The remaining @-gal positive colonies (48%+38) grew as dark blue colonies with large blue haloes (2 + +). Subcultures and identifications were performed on 78 colonies; all strains belonged to the genus Bifidobacterium. In order to evaluate the ability of the X-gal medium to enumerate Bifidobacterium, we compared counts of positive colonies (Z + +) on the X-Gal medium with counts on the selective medium (MB) described by Beerens [lo] (Fig. 1). For 11 of the 13

Colonies + + / + + f

9.12 3.39 0.85 3.09 3.63

according according

to the analysis

(39.8) ( 100.0) (14.8)

22.9 I 3.39 5.15 3.09 5.13

calculated calculated

3.3. Application of the procedure of faecal samples

(%y

1 2 3 4 5

“Percentages “Percentages

small diffusion zone surrounding them. Of five strains of Lactobacillus tested, only one gave a blue ring (LB4). Clostridium pevfringens, Escherichia coli, Enterococcus, Streptococcus, and Peptostreptococcus did not produce blue haloes.

+I++/+++

( 100.0)

(100.0)

C. Favier et al. I Journal of Microbiological Methods 27 (1996) 25-31

0

s-

0

‘l‘2

9

8

X-gal Fig. 1. Correlation between the counts of bifidobacteria as the log” cfu g-’ wet weight of faecal material.

29

I

10

medium

growing on MB medium and the counts on X-gal medium. The results are expressed

samples studied, the two counts correlated well (Y= 0.9). In two samples, the number of bifidobacteria was underestimated using the MB medium because Bifdobacterium colonies were partially masked by high numbers of Gram positive rods without typical bifidobacterial morphology (516 for one sample, 8/9 for the second).

4. Discussion X-gal as chromogenic substrate for &gal is commonly used for the detection of coliforms under aerobic conditions [ 111. Upon hydrolysis, this compound releases an indoxyl derivative which is quickly oxidized to insoluble indigo. The latter remains localized at the site of the enzyme’s presence. Therefore, positive colonies are easily visualized and can be further sulbcultured. Chevalier et al. [7] previously describe’d a X-a-gal based medium for selective enumeration of bifidobacteria. Plates were incubated for 48 h under anaerobic atmosphere containing N, 85%, H, lo%, CO, 5%. A subsequent exposure to aerobc atmosphere for 6-8 h was necessary to provide the blue staining. With our X-gal medium, the anaerobic atmosphere generated either by GasPack or AnaeroGen systems did not permit the staining of the P-gal positive strains. However, we demonstrated that the development

of the colour in anaerobic conditions is possible by incubation either in an anaerobic chamber (N2 75%, H, lo%, CO, 15%) or using the Generbag system. Because the colour is developed in anaerobiosis, the colonies can be subcultured and identified without injury. Additional information on the composition of the atmosphere provided by the different commercial systems available might help to explain the observed discrepancies. We checked for the absence of oxygen in these systems by using methylene blue test strips as indicator. X-gal gives an insoluble coloured hydrolysis product. This allows to visualize colonies such as Clostridium strains producing intracellular P-gal which are revealed by a blue undiffused colour. This was also true for one E. coli strain; surprisingly the second remained negative, which might be due to either the absence of an inducer in the culture medium or lack of a permease allowing the penetration of the substrate into the bacterial cell. Other colonies release P-gal into the culture medium. In this case, the blue diffuse zone might result either from a secretion of the enzyme by bacterial colonies or a release of intracellular or cell-associated P-gal occurring during bacterial lysis. Irrespective of the mechanism, the obtained patterns make the further identification of bacterial strains producing P-gal easier. We tested different strains belonging to the genus

30

C. Favier et al. I Journal of Microbiological Methods 27 (1996) 25-31

Bijidobacterium on the X-gal medium. All the strains of B. bifidum and B. breve exhibited strongly positive responses. The other species of B$dobacterium were indicated by a blue ring, more variable in size. The proportionality between the size of the ring and the P-gal activity released from those colonies has not been established, although Hoskins et al. [12] already reported that, among the strains able to degrade mucins, B. bifidum produces a more important extracellular /?-gal activity than B. infantis and B. longum. Moreover, as recently reported, [13], &gal is cell-associated in B. longum. Hoskins [14] reported that strains of Ruminococcus torques secreted larger amounts of P-gal than strains of R. gnavus. In agreement with these data, we also observed that strains of R. lactaris produced larger blue rings than Ruminococcus gnavus. Bacteroides exhibited small blue zones (+ ); since glycosidases produced by Bacteroides are predominantly cell-bound [14-161, the small haloes surrounding the colonies might be explained by the release of cell-bound /?-gal by aging bacteria. Salyers et al. [17] showed that lactobacilli were not able to ferment polysaccharides. In our study, only one strain of Lactobacillus among the five tested formed a blue zone surrounding the colonies. The strains of Enterococcus, Streptococcus sp., and Peptostreptococcus we studied, did not produce P-gal activity. These data are in good agreement with previous results of Hoskins et al. [12] who reported that one strain of Enterococcus faecalis was unable to produce p-gal activity. The X-gal medium was further applied to the analysis of faecal flora. The colonies with a very small blue zone surrounding them (51 mm) were predominant and mainly identified as Bacteroides. The mean number of these bacteria was near lo9 g-’ of wet faeces. Ruminococcus spp. are known to constitute only a minor part of the faecal flora [12]. We did not find species of this genus during our analysis of faeces. All strains with large ring diameters (2 + +) were identified as bifidobacteria, which thus appear, to a large extent, to be responsible for the soluble /?-gal activity present in faecal extracts. Their count was comprised between 10’ and lo9 g-’ of faeces. We noticed that pure strains of Bijidobacteria frequently exhibited small rings (+) whereas strains belonging

to the same genus but isolated from faecal samples showed larger ones (2 + +). Thus BiJidobacteria might express higher P-gal activity in natural samples than pure strains currently subcultured on lactose-free medium. A good correlation was observed when counts of colonies with blue rings (2 + +) were compared to those obtained onto MB medium, made more specific for bifidobacteria by Gram staining. However, with the MB medium, counts of Bifidobacterium colonies were underestimated in two samples. This observation might be explained by the occasional presence of a high level of interfering bacteria growing faster than the bifidobacteria on the selective medium. The procedure we propose seems to be suitable for the study of faecal bacteria releasing P-gal in the culture media. It allows to select the colonies producing this enzyme from a complex anaerobic flora. Applied to the analysis of faeces from patients with acute CD, it might help to understand the mechanism leading to the decrease of faecal P-gal activity observed in these patients. Studies in this respect are now in progress in our laboratory. Furthermore, the new methodology we propose might find other applications including analysis of dairy products, water. . . , and characterization of anaerobic bacteria producing all of the enzymes able to release bromo-chloro-indoxyl from their specific substrates. This chromogenic method, which does not involve time-consuming isolation procedures and enzyme assays on isolated strains, makes the detection, within a complex flora, of bacteria species able to specifically produce an enzyme activity easier and faster.

Acknowledgments This work was supported in part by the Minis&e de 1’Enseignement Superieur et de la Recherche, by the University of Lille II (contrat DRED-E.A. 1052) and by a grant from the Laboratoires Biocodex and the Conseil Regional du Nord-Pas-de-Calais. We are indebted to Dr. Frangoise Gavini (INRA, Villeneuve d’Ascq, France) for the gift of the reference strains and helpful discussions, Prof. Henri Beerens for his

C. Favier

et al. I Journal of Microbiological Methods 27 (1996) 25-31

expert judgment, Noelle Cartegnie and Laurent Daels for skillful assistance.

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