Ann. Mierobiol. (Inst. Pasteur) 1983, 134 A, 339-349

A N T I M I C R O B I A L S U S C E P T I B I L I T Y

OF B I F I D O B A C T E R I U M

by D. Matteuzzi 0), F. Crociani (~) and P. Brigidi (0

(1) Cattedra di Chimica delle Fermentazioni e Microbiologia Industriale, FacoltO di Farmacia, and

(2) Istilulo di Microbiologia Agraria e Tecnica, Facoll~ di Scienze Agrarie, Universildt degli Studi di Bologna, Bologna (Italy)

SUMMARY

The susceptibility pattern of 459 strains of bifidobacteria, representing 15 species, to 16 antimicrobial agents was determined by the broth dilution method. The majority of the strains derived from human faeces. Penicillin G, erythromycin, clindamycin, vancomycin and bacitracin were the most active compounds; they inhibited 90 % of the strains at less than 1.6 ~g/ml. All strains were susceptible to chloramphenicol (MICro----2.0-5.8 ~g/ml) and also to lincomycin (MICso =- 0.64-1.5 ~g/ml). Neomycin, streptomycin and tetracycline presented a great variability in their activity. Most strains were resistant to polymyxin B, nalidixic acid, kanamycin, gentamicin and metronidazole. The only variation in susceptibility which was observed among the different species concerned Bifidobaclerium suis, which generally appeared to be more resistant than other species.

KEY-WORDS: Bifidobacterium, Antibiotic susceptibility, Anaerobic flora; Pathology, Therapy, Intestinal equilibrium, Diagnosis.

INTRODUCTION

It is well known that the administration of antimierobial substances to man may cause the suppression of some intestinal bacterial groups such as bifidobacteria, lactobacilli, etc., which positively affect natural resistance to infection. The altered microbial balance induces intestinal disorders, especially pseudomembranous colitis and other non-specific colitis [7, 8, 12].

The determination of the susceptibility of normal gastrointestinal flora to various antimierobial agents would provide the clinician with more spe-

Manuscrit re~u le 21 janvier 1983, accept~ le 5 avril 1983.

340 D. MATTEUZZI, F. CBOCIANI AND P. BR1GIDI

cific information for guidance in therapy. Since bifidobacteria consti tute a major component of human and animal intestinal flora, especially in the new-born, it is of great interest to know their antimicrobial suscepti- bil i ty pattern. Li terature on the antimicrobial susceptibil i ty of bifidobac- teria is not abundant [1, 2, 9, 13, 16, 17, 23], and generally only a limited number of species and strains have been taken into consideration. For this reason, the aim of the present s tudy was to assay the in vitro effect of seve- ral drugs, some of which are currently in use for therapy in anaerobic infections, against 459 strains of bifidobacteria isolated from various habi- ta ts and, in particular, from human faeces.

M A T E B I A L S AND M E T H O D S

Bacterial slrains.

A total of 459 bifidobacteria strains were tested. All organisms were isolated from various habitats such as human faeces (adult and infant), faeces of various animals, human vagina, dental caries, sewage and cattle rumen (table I) and were maintained in the culture collection of this Institute.

TABLE 1. - - List of strains and their sources.

Nb of s t ra ins Species examined Source

B. breve 89 H u m a n faeces B. bifidum 80 H u m a n faeces B. longum 80 H u m a n faeces B. adolescentis 70 H u m a n faeces B. infantis 50 H u m a n faeces B. suis 20 Piglet faeces

Sewage B. calenulatum 1 3 ~ Adu l t faeces

j 7 Cat t le r u m e n B. lhermophilum 11~..4 Pig faeces

Den ta l caries B. dentium 8 ~ Adul t faeces B. indicum 8 Bee in tes t ine B. asteroides 7 Bee in tes t ine B. corgne[orme 7 Bee in tes t ine B. globosum 7 Cat t le r u m e n

Sewage B. angulatum 6 ~ Adul t faeces

1 Pig faeces B. pseudolongum 3 ~ 1 Chicken faeces

Mouse faeces

Media and culture conditions.

The liquid medium TPY [19] was used in this study, since it supported the best growth of all bifidobacteria [19]. Mueller-Hinton broth (BBL) was used in certain comparative studies, since it is the medium in which standard MIC of antibiotics

MIC = min i ma l inh ib i tory concen t ra t ion .

ANTIMICROBIAL AGENTS AND BIFIDOBACTERIUM 341

are generally determined. Anaerobic conditions for the cultivation of organisms and susceptibility testing were obtained by employing the ~ Anaerobic Glove Box ~ (Forma Scientific, rood. 1024, Ohio, USA).

Antimierobial agents and susceptibility testing.

Beferenee powders for susceptibility testing obtained from Sigma (Saint-Louis, Mo., USA) were weighted, dissolved and diluted in appropriate diluents, and filter- sterilized prior to addition to TPY medium. The following antimicrobial agents were used: penici|lin G, tetracycline, chloramphenicol, erythromycin, bacitraein, neomycin, streptomycin, kanamycin, polymyxin B, gentamicin, vancomycin, lineomycin, clindamycin, metronidazole, nitrofurantoin and nalidixic acid. Mini- mal inhibitory concentrations (MIC) were determined by using a broth dilution method. An inoculum of 105-10 ~ organisms per milliliter was used. The tubes, incu- bated at 37 ~ C for 48 h, were made in triplicate and the MIC was the least concen- tration of compound which prevented visible growth.

MIC were subjected to correlation and linear regression analysis. MICro was the lowest concentration of antibiotic which inhibited at least 50 % of the strains tested, and MICgo was the lowest concentration which inhibited at least 90 % of the strains tested.

In the first part of the study, a survey of the antimicrobial susceptibility of 15 species of bifidobacteria isolated from different habitats was performed. During the second part of the study, the five species of bifidobacteria commonly found in the human alimentary tract were tested for their susceptibility to five antimicrobials (penicillin G, clindamycin, lincomycin, vancomycin and metro- nidazole) generally used against anaerobic infections, and to gentamicin, whose activity against bifidobacteria has not yet been assayed.

RESUL T S

The act ivi ty of penicillin G, erythromycin, bacitracin, chloramphenicol, tetracycline and nitrofurantoin against 15 species of the genus Bifidobacte- rium are summarized in table II. Penicillin G (also see table IV) and ery- thromycin were the most active compounds, having an MICro in the range of < 0.05-1.6 ~g/ml. Bacitracin was slightly less active (MICgo ---- 0.05- 1.4 units/ml). Among all species studied, B. suis showed the lowest suscep- t ibi l i ty to these antibiotics.

The broad-spectrum antibacterial agents chloramphenicol and nitro- furantoin behaved similarly, with their MIC included in a narrow range of 2.0-5.8 and 13-73 ~g/ml, respectively; tetracycline showed a wide range of MICro (3-238 ~g/ml). Table III shows the susceptibil i ty of bifidobacteria to some aminoglycosides, to polymyxin B and to nalidixic acid. The first three compounds presented a very low activity, and kanamycin appeared to be the least active: its MICgo was always > 130 ~g/ml. The species B. bifidum and B. suis seemed to be those most resistant to kanamycin and neomycin. Compounds with a Gram-negative spectrum, such as nalidixic acid and polymyxin B, showed a low act ivi ty against bifidobacteria. Nali- dixic acid inhibited 90 % of bifidobacteria at 344-777 ~g/ml, and poly- myxin B at > 1,000 ~g/ml.

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Table IV reports the results of the second part of the study concerning the susceptibility of the five most common species of human gastrointestinal tract bifidobacteria to some antimicrobial agents employed in anaerobic infections and to gentamicin. The most active compounds were penicillin G and clindamycin, which inhibited the growth of 90 % of the strains studied at coucentrations ranging from 0.09 to 0.89 ~g/ml. Vancomycin also appeared to be very active, having an MICg0 of 0.73-0.85 ~g/ml, whereas lincomycin required slightly higher concentrations (MICgo = 1.4-5.8 ~g/ml). Almost all species were resistant to metronidazole (MICgo = > 500 ~g/ml) and gentamicin (MICgo = 104-> 500 ~g/ml), with the exception of a few strains of B. infantis susceptible to 1.5-3 ~g/ml of this latter compound.

DISCUSSION

A prerequisite for a complete understanding of how antimicrobial agents induce intestinal disorders is the assessment of their effects upon normal gut flora. Our results may contribute to this knowledge, since they concern a vast number of bifidobacteria which represent one of the most prevalent bacterial groups. Literature on the effects of many antibacterial agents upon Bifidobacterium in vitro is very abundant [4, 5, 6, 10, 11, 14, 20, 21, 22] but the true role of bifidobacteria in various physiological and pathophysiological processes affecting the host has not been defned [ 17, 18].

Our data concerning antimicrobial susceptibility at the genus level (15 species examined) show that penicillin G, erythromycin, chloramphe- nicol and bacitracin were the most active compounds.

Most of the species are also sensible to nitrofurantoin, whereas tetra- cycline shows great variability in its activity. Resistance to kanamycin, neomycin, streptomycin (except for B. asteroides), nalidixic acid and poly- myxin B with respect to achievable serum levels of antibiotics is constant in the genus. These results confirm the well-known natural resistance of anaerobes to aminoglycosides [15]. Among the species examined, only B. suis shows less susceptibility to various antimicrobial agents.

Many antimicrobials used in this study have been previously assayed for their activity against bifidobacteria of human origin by Miller and Finegold [17] and Sutter and Finegold [23]; their results are in almost com- plete agreement with ours for the same species tested.

Antimicrobials currently used against anaerobic infections are very active against bifidobacteria; this suggests a consistent elimination of this bacterial group from the faecal flora [4, 22].

In terms of susceptibility to metronidazole, we found a higher degree of resistance on the part of bifidobacteria compared to other data [2, 23]. Therefore, this compound, as suggested by Essers [3], may be used for iden- tification of the genus. As concerns the use of antibiotics as selective media for bifid bacilli, the addition to the medium of 80 ~g/ml of kanamycin is suggested.

348 D. MATTEUZZI, F. CROCIANI AND P. BRIGIDI

13]~SUMt~

SENSIBILITI~ DES BIFIDOBACTERIES AUX ANTIMICROBIENS

Nous avons ~tudi~ la sensibilit~ de 459 souches de Bifidobaclerium appar tenant ~ 15 esp~ces : la CMI de 16 agents antibact6riens a ~t~ 6valu6e par la m6thode des dilutions en milieu liquide. Les bifidobact~ries se sont r~v~l~es tr~s sensibles 5 la p~nicilline G, ~ l '~rythromycine, ~ la elindamycine,

la vancomycine, au chloramph~nieol et ~ la bacitracine. La n6omycine, la s t reptomycine et la t6tracycline pr~sentent une activit~ tr~s variable. La plupart des souehes se sont montr6es r~sistantes /~ la polymyxine B, l 'aeide nalidixique, ~ la kanamycine, ~ la gentamicine et au m~tronidazole. Seul B. suis semble ~tre moins sensible que les autres esp~ces aux substances 6tudi~es.

MOTS-CLt~S : Bifidobacterium, Antibiosensibilit6, Flore ana6robie ; Patho- logic, Traitement, t~quilibre intestinal, Diagnostic.

ACKNOWLEDGEMENTS

We thank Andrea Guglielmi for his assistance in the computer handling of much of this information.

REFERENCES

[1] APPELBAUM, P. C. & CHATTERTON, S. A., Susceptibility of anaerobic bacteria to ten antimicrobial agents. Anlimicrob. Agents a. Chemother., 1978,

[21 CHOW, A. W., PATTEN, V. & GUZE, L. B., Susceptibility c,f anaerobic bacteria to metronidazole: relative resistance of non-spore-forming Gram-positive bacilli. J. in/eel. Dis., 1975, 131, 182-185.

[3] ESSERS, L., Simple identification of anaerobic bacteria to genus level using typical antibiotic susceptibility patterns. J. appl. Bact., 1982, 52, 319-323.

[41 FINEGOLD, S. M., HARADA, N. E. & MILLER, L. G., Lincomycin: activity against anaerobes and effect on normal human fecal flora. Antimicrob. Agents a. Chemolher., 1965, n o 5, 659-667.

[5] FINEGOLD, S. M., MILLER, A. B. ~ POSNICK, D. J., Further studies on selective media for Bacleroides and other anaerobes. Erni~hrungs[orschung, 1965, 10, 517-528.

[6] FINEGOLD, S. M., SUTTER, V. L., BOYLE, J. D. & SHIMADA, K., The normal flora of ileostomy and transverse colostomy effluents. J. in/ect. Dis., 1970, 122, 376-381.

[7] GEDDES, A. M., SLEET, R. A. & MURDOCH, J. McC., Lincomycin hydrochloride: clinical and laboratory studies. Brit. reed. J., 1964, 2, 670-672.

[8] GEORGE, W. L., KIRBY, B. D., SUTTER, V. L. & FINEGOLD, S. M., Antimicrobial susceptibility of Clostridium difficile, in ~ Microbiology, (S. Schlessinger) (p. 267-271). American Society for Microbiology, Washington, 1979.

[9] GUILLERMET, F. N. & COURTIEU, A. L., t~tude sur la sensibilit6 aux anti- biotiques de 164 Asporulales et 50 Actinomyc6tales anadrobies. Ann. Ann. Inst. Pasteur, 1966, 111, 503-512.

ANTIMICROBIAL AGENTS AND B I F I D O B A C T E R I U M 349

[10] HAENEL, H., KOHLER, F., MERTSCH, H. & PARDEMANN, C., Uber den Einfluss yon Tetracyclinen auf die faekale Microbiozonose yon TPE Daueraus- scheidern. Zbl. Bakt., I. Abt. Orig., 1960, 177, 41-61.

[11] HAENEL, H., MERTSCH, H., KOHLER, F. & MfiLLER-BEUTHOW, W., Verfinde- ruugen der faekalen Mikrofora bei Jugendlichen durch Paromomycin. Zbl. Bakl., I. Abt. Orig., 1965, 195, 385-392.

[12] HOLLOWAY, W. J., KAHLBAUGH, R. A. & SCOTT, E. G., Lincomycin: a clinical study. Anlimierob. Agents a. Chemother., 1964, n ~ 4, 200-203.

[13] HUBERT, Th., LAUBE, M., SCHOUTENS, E. & YOURASSOWSKY, E., I~tat actuel de la sensibilit6 des bact~ries anadrobies aux antibiotiques. Rev. Insl. Pasteur Lyon, 1981, 14, 217-224.

[14] KNOTnE, H. & WIEr)EMANN, B., Die Wirkung yon Ampicillin auf die Darm- flora des gesunden Menschen. Zbl. Bakl. I. Abt. Orig., 1965, 197, 234-243.

I15[ LAMBE, D. W. Jr & LASLIE, W. W., A standardized disk method for anti- biotic susceptibility testing for anaerobes; four years of antibiotic data, in (( Anaerobic bacteria. Selected topics )). (D. W. Lambe, Jr, R. J. Genco & K. J. Mayberry-Carson) (p. 51-60). Plenum Press, New York, 1980.

[16] DE LAVERGNE, E., BURDIN, J. C., SCHMITT, J. & MANCIAUX, M., Sensibilit6 de Bifidobacterium bifidum fi onze antibiotiques. Ann. Inst. Pasteur, 1959, 97, 104-107.

[17] MILLER, L. G. & FINEGOLD, S. M., Antibacterial sensitivity of Bifidobacterium (Lactobacillus bifidus). J. Bact., 1967, 93, 125-130.

[18] POUPARD, J. A., HUSAIN, I. & NORRIS, lq. F., Biology of the bifidobacteria. Bact. Bey., 1973, 37, 136-165.

[19] SCARDOVI, V., The genus Bifidobacterium, in (( The Prokaryotes. A handbook on habitats, isolation and identification of bacteria )) (P. S. Mortimer, H. Stolp, H. G. Trfiper, A. Balows & H. G. Sehlegel) (p. 1951-1961). Springer-Verlag, Berlin-Heidelberg, 1981.

[20] SCHABINSKI, G. & ALTHAMMER, R., Untersuchungen fiber die Darmwirksamkeit yon Streptomycin. Klin. Wschr., 1958, 36, 1013-1017.

[21] SEELmER, H. P. R., Die Wirkung von Erythromycin auf die Darm-Flora des gesunden Menschen. Arzneimitlel-Forsch., 1957, 7, 629-633.

[22] SUTTER, V. L. & FINEGOLD, S. M., The effect of antimierobial agents on human fecal flora: studies with cephalexin, cyclacillin and clindamycin, in (( The normal microbial flora of man )) (F. A. Skinner & J. G. Carr) (p. 229-240). Academic Press, London, New York, 1974.

[23] SUTTER, V. L. & FINEGOLD, S. M., Susceptibility of anaerobic bacteria to 23 antimicrobial agents. Antimicrob. Agents a. Chemolher., 1976, 1O, 736-752.