INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF CERTAIN
VETERINARY DRUG RESIDUES IN FOOD
WHO FOOD ADDITIVES SERIES 41
Prepared by:
The 50th meeting of the Joint FAO/WHO Expert
Committee on Food Additives (JECFA)
World Health Organization, Geneva 1998
TETRACYCLINES: OXYTETRACYCLINE, CHLORTETRACYCLINE, AND
TETRACYCLINE(addendum)
First draft prepared by
Dr H. Fernandez and Dr M. Miller
US Food and Drug Administration
Washington DC, USA
1. Explanation
2. Biological data
2.1 Absorption, distribution, and excretion
2.2 Effects on enzymes and other biochemical parameters of the
intestinal flora
2.3 Microbiological studies
2.3.1 In vitro
2.3.1.1 Minimal inhibitory concentration
2.3.1.2 Continuous flow chemostat system inoculated
with tetracycline
2.3.1.3 Effect of tetracyclines on colonization
resistance
2.3.1.4 Selection of bacterial strains resistant to
tetracycline
2.3.2 Animal models
2.4 Studies in humans
3. Comments
4. Evaluation
5. References
1. EXPLANATION
The tetracyclines oxytetracycline, chlortetracycline, and
tetracycline are drugs with a long history of use. These products were
first evaluated as a group at the twelfth meeting of the Committee
(Annex 1, reference 17), and a temporary ADI of 0-0.15 mg/kg bw was
established. Oxytetracycline was reevaluated at the thirty-sixth
meeting of the Committee (Annex 1, reference 91), which concluded
that:
* The absorption of oxytetracycline in humans after oral
administration is greater than in mice and pigs (about 60%
compared with 4-9%). After absorption, oxytetracycline is widely
distributed in the body and eliminated mainly in the urine as
parent drug.
* There was no evidence that oxytetracycline is carcinogenic in
mice or rats. Toxicity was observed in experimental animals only
at doses higher than those that caused microbiological effects.
The lowest NOEL in the toxicological studies in animals was 18
mg/kg bw per day.
* The NOEL for the induction of resistant coliforms in the
intestinal microflora in a six-week study in dogs was 0.05 mg/kg
bw per day, while in humans the NOEL was 2 mg/person per day,
equivalent to 0.03 mg/kg bw per day. With a safety factor of 10
to account for variation in the intestinal microflora of humans,
an ADI of 0-0.003 mg/kg bw was established. The next highest dose
in the study in humans was 20 mg/day.
Chlortetracycline and tetracycline were reevaluated at the
forty-fifth meeting of the Committee (Annex 1, reference 119), which
concluded that:
* In humans receiving oral therapeutic doses, 30% of the dose of
chlortetra-cycline was absorbed, as compared with 60-80% of an
oral dose of tetracycline. The absorption is impaired by divalent
cations such as calcium and magnesium, which form complexes with
the drug. Tetracycline is excreted mainly in the urine, and
chlortetracycline is excreted in urine and faeces. Both drugs are
widely distributed in the body, the highest concentrations of
residue being found in kidney and liver.
* Tumour incidences were not increased in mice or rats treated with
tetracycline or chlortetracycline.
* Tetracycline and chlortetracycline did not have significant
reproductive or developmental effects, and induced general
toxicity in experimental animals only at high doses. Data on the
reproductive toxicity of tetracycline were not available.
* The geometric mean MICs for chlortetracycline and oxytetracycline
against selected microorganisms of the human intestinal
microflora are similar.
* The Committee concluded that antimicrobial activity is the most
sensitive end-point for determining the ADI for tetracycline and
chlortetracycline. A group ADI of 0-0.003 mg/kg bw was
established for oxytetracycline, tetracycline, and
chlortetracycline separately or in combination because of their
similar antimicrobial activity. This ADI is based on the NOEL of
2 mg/person per day determined in a study in humans with
oxytetracycline that was reviewed at the thirty-sixth meeting.
The Committee noted that this ADI provides an adequate margin of
safety when compared with the lowest NOEL for toxicological
effects of 100 mg/kg bw per day for chlortetracycline in dogs.
At the tenth session of the Codex Committee on Residues of
Veterinary Drugs in Foods, the Expert Committee was requested to
reconsider its previous evaluation of the tetracycline group.
2. BIOLOGICAL DATA
2.1 Absorption, distribution, and excretion
The tetracyclines are incompletely absorbed from the human
gastrointestinal tract; 30% chlortetracycline and 60-80%
oxytetracycline and tetracycline are absorbed from an empty stomach.
When humans were treated orally with 1 g/d of tetracycline, the
concentration in the faeces was 10-97 µg/g (Van Marwyck, 1958).
2.2 Effects on enzymes and other biochemical parameters of the
intestinal flora
Because the tetracyclines are incompletely absorbed from the
gastrointestinal tract, high concentrations are readily achieved in
the intestine, which perturb the intestinal microflora within 48 h of
daily treatment. Both therapeutic and subtherapeutic doses of
tetracyclines can perturb the intestinal microflora by inducing
resistant strains, altering the metabolic activity and the
colonization resistance of the flora (barrier effect) to allow
overgrowth of pathogenic, opportunistic, or resistant microorganisms,
and altering the ecological balance of the flora with no identified
deleterious effect (Corpet & Brugère, 1996).
The tetracyclines have similar microbiological activity and are
considered to be 'broad spectrum' antibiotics. Although these drugs
were originally very active against gram-positive bacteria, many of
those bacteria have become resistant to tetracyclines, and many
gram-negative bacilli have also become less susceptible. Anaerobic
bacteria that are resistant to the tetracyclines have also emerged.
The bacteriostatic effect of the tetracyclines is due to inhibition of
bacterial protein synthesis. They bind to 30S subunits of bacterial
ribosomes and prevent attachment of aminoacyl-tRNA to the ribosomal
receptor site, thus altering protein synthesis and therefore cell
multiplication. The inhibitory effect of tetracyclines is usually
reversible when the drug is removed. Although tetracycline appears to
have a greater effect on the selection of resistant bacterial strains
than the other tetracyclines, microorganisms that are resistant to one
of the tetracyclines are frequently resistant to the other compounds
in this class (Brown, 1988; Sande & Mandell, 1990; Chopra et al.,
1992; Roberts, 1996).
Alterations in the metabolism of the intestinal microflora have
been demonstrated in human volunteers given therapeutic doses of
oxytetracycline. The drug caused a marked reduction in the
esterification and transformation of faecal neutral sterols, which are
biochemical processes carried out by the intestinal microflora
(Korpela et al., 1984). Oxytetracycline at therapeutic doses has also
been shown to increase faecal concentrations of conjugated oestrogens
in men, probably due to the decreased hydrolytic effect of
beta-glucuronidase produced by the intestinal microflora (Hamalainen
et al., 1987). Women receiving the birth control drug
ethyniloestradiol and treated with tetracycline had a markedly
decreased average circulating half-life of the oestrogen and increased
faecal excretion of ethyniloestradiol due to decreased intestinal
reabsorption of the oestrogen. Administration of tetracycline reduced
the beta-glucuronidase activity of the microflora and therefore
altered the metabolism of oestrogens (Gorbach, 1993). When therapeutic
doses of tetracycline were given to healthy volunteers who were
subsequently treated with salicylazosulfapyridine, a drug used in the
treatment of ulcerative colitis, the metabolism of
salicylazosulfapyridine was altered. While untreated humans excrete
the break-down products, the subjects given tetracycline excreted
salicylazosulfapyridine intact in the faeces, indicating a reduction
in bacterial azoreductase activity (Gorbach, 1993). It has also been
demonstrated that the metabolism of digoxin, a cardioglycoside that is
normally metabolized in the intestine, is altered by the action of
antibiotics on the intestinal microflora. The metabolic activity of
the microflora, mainly the anaerobic bacteria Eubacterium lentum, is
responsible for reduction of the lactone ring and hydrolysis of the
glycoside moiety. Tetracycline therapy decreases urinary excretion of
the digoxin reduction products, which can result in an increased serum
concentration of digoxin and may cause digitalis-induced toxicity
(Lindenbaum et al., 1981).
Diarrhoea and superinfections are common side-effects that limit
the therapeutic use of tetracyclines. Tetracyclines inhibit the growth
of many aerobic and anaerobic coliform microorganisms and
gram-positive spore-forming bacteria. As the faecal coliform count
decreases, tetracycline-resistant organisms, particularly yeasts and
the enterococci Proteus and Pseudomonas, overgrow, and
superinfections can develop. Of the superinfections that occur as a
result of tetracycline therapy, antibiotic-induced diarrhoea and
pseudomembranous colitis due to a cytotoxic toxin produced by the
overgrowth of Clostridium difficile are the most important in
humans. The incidence of gastrointestinal distress increases with
dose. In general, the normal intestinal flora is restored several days
after treatment is withdrawn (Nord et al., 1984; Sande & Mandell,
1990; Ramos et al., 1996).
2.3 Microbiological studies
2.3.1 In vitro
When Staphylococcus aureus ATCC 9144 was exposed to 30 ppb
oxytetracycline for 14 days, the MICs of oxytetracycline were similar
to those of bacteria that were not exposed to the antibiotic,
suggesting that 30 ppb is not sufficient to select for resistant
bacteria in vitro. When oxytetracycline was combined with low
concentrations of other antimicrobials (neomycin, erythromycin,
sulfamethazine, and/or dihydrostreptomycin), the MIC increased (Brady
et al., 1993). Similar results were obtained by Brady and Katz (1992)
when they studied the effect of low doses of antibiotics on
Staphylococcus aureus ATCC 9144 and Enterobacter cloacae B520
after 14 days of incubation. These data showed that a combination of
antimicrobials can cause a decrease in bacterial sensitivity.
In an investigation of the ability of low concentrations of
antimicrobials to affect the conjugal transfer of resistance among
Escherichia coli strains, oxytetracycline added at a concentration
of 0.01, 0.1, or 1.0 µg/L to flasks containing recipient and donor
E. coli strains did not increase the transfer of resistant plasmids.
Oxytetracycline at 0.01 µg/L, however, decreased the transfer of
resistant plasmids (Brady & Katz, 1988). In a similar study, the
development of resistance was studied with E. coli CS-1 (a chicken
faecal strain) and Enterobacter cloacae B520 (a human intestinal
strain). Both organisms showed decreased susceptibility (measured by
the MIC) to chlortetracycline and oxytetracycline at concentrations of
0.1œ5 µg/L. No change in susceptibility was seen with other
antibiotics (streptomycin, tylosin, sulfamethazine, bacitracin,
virginiamycin, flavomycin, and monensin) at a concentration of 1 µg/L.
The results varied markedly, however, according to the strain used and
the method for measuring resistance (Brady et al., 1988).
In a study with a continuous flow chemostat model system
containing an E. coli culture and subinhibitory concentrations of
antibiotics, 0.25 µg/ml (0.25 mg/L) of tetracycline resulted in
selection of an R-plasmid strain that grew faster than the sensitive
strain (Egger & Lebek, 1985). Interpretation of this study was
difficult, because the culture did not contain species representative
of human intestinal microflora.
2.3.1.1 Minimal inhibitory concentration
MICs were determined with four strains of each of the 50 most
numerous species in the human colon by the method recommended by the
National Committee for Clinical Laboratory Standards (NCCLS) for
anaerobic bacteria. The tests were performed in anaerobiosis, at pH
6.0, 6.5, or 7.0, with inoculum densities of 105 and 107 cells per
spot. The bacteria used included strains isolated from normal human
intestinal microflora over a period of about 30 years. The results for
the Bacteroides species showed that their resistance has increased
over the years. Strains with MICs for tetracycline < 0.5 µg/ml were
isolated between 1933 and 1969; strains with MICs > 1 µg/ml were
isolated after 1970. These results cannot readily be compared with
those for more recently isolated bacterial strains because the
susceptibility of strains isolated several years ago differs markedly
from that of strains isolated today.
The MICs of 10 strains of each of 10 species of human colonic
bacteria (E. coli, Bifidobacterium spp., Bacteroides fragilis,
Eubacterium spp., Clostridium spp., Streptococcus spp.,
Fusobacterium spp., Lactobacillus spp., Proteus spp., and
Peptostreptococcus spp.) and three reference strains (E. coli
7624, B. fragilis 7716 T, and C. perfringens 6019) were
determined. All of the bacterial species were isolated from healthy
volunteers in 1992 and 1993. The MIC50, MIC90, and geometric MIC
were calculated for each bacterial species and each antimicrobial
agent, and the sensitivity of the bacterial strains to oxytetracycline
and tetracycline was compared. The concentrations of these drugs that
had no effect on any of the 10 strains were also presented. In
general, the MIC50, the MIC90, the geometric mean, and the
concentrations of oxytetracycline and tetracycline that had no
inhibitory effect on bacterial growth were very similar; however,
tetracycline was more active against Bifido-bacterium spp.,
Eubacterium spp., and Fusobacterium spp. and less active against
Streptococcus spp. The geometric mean MIC for all the tested strains
(with a value of 32 µg/ml given to resistant strains) was very similar
for oxytetracycline (3.81 µg/ml) and tetracycline (3.25 µg/ml). The
most sensitive relevant genera were Fusobacterium spp. and
Clostridium spp., with an MIC50 of 0.2 µg/ml (Richez, 1994).
MICs were also determined by the Centre National d'Etudes
Vétérinaires et Alimentaires (1997) in France (Table 1). The tests
were performed with bacterial strains isolated from the faeces of
human volunteers and tested by the NCCLS method. In general, aerobic
and anaerobic bacteria were susceptible to tetracycline, i.e. more
than 50% of the strains were inhibited below the break-point value of
8 µg/ml. A large number of aerobic strains (Enterococcus and
E. coli) isolated in brainœheart infusion agar and enterococcus
media had an average MIC50 of 1 µg/ml. A few strains of E. coli
isolated aerobically in McConkey medium had an average MIC50 of
8 µg/ml, but their MIC90 was much higher (64 µg/ml). Anaerobic
strains had an average MIC50 of 1 µg/ml in both blood-supplemented
brainœheart infusion agar and Schaedler media. Gram-positive anaerobic
flora were more susceptible to tetracycline than gram-negative flora.
In general, the results of this study are very similar to those
obtained by Richez (1994).
2.3.1.2 Continuous flow chemostat system inoculated with tetracycline
In a series of experiments carried out by Techlab, Inc. (1997),
single-chambered chemostats with continuous flow were inoculated with
a pool of faeces from healthy volunteers and maintained under
conditions mimicking the human colon. The medium used in the chamber
contained elements found in omnivorous diets (casein, peptone, and
complex carbohydrates from plants) supplemented with bile, L-cysteine,
salts, haemin, vitamins, and Tween 80. The lot of medium in the
control chemostat (no drug) was changed at the same time as that in
the test chemostats. After inoculation with faeces, the chemostat
culture was allowed to reach steady-state (generally at about two
weeks), which was confirmed by measuring the following
microflora-associated characteristics: (i) general microscopic
appearance of the culture fluids; (ii) cellular fatty acids; (iii)
molar ratio of short-chain fatty acids; (iv) sulfate reduction; (v)
counts of target organisms within the normal flora, including
facultative coliforms and enterococci; (vi) bacterial enzymes
including beta-glucosidase, beta-glucuronidase, nitroreductase, and
azoreductase; and (vii) bacterial metabolism of bile acids. When
steady-state was reached, each chemostat was inoculated with 0
(control), 0.15, 1.5, or 15 mg/ml of tetracycline. These doses
correspond to ADI values of 0, 0.025, 0.25, and 2.5 mg/kg bw, assuming
that about 100 µg of tetracycline are present in 1 g faeces or colon
Table 1. Susceptibility to tetracylcine of identified anaerobic strains isolated from faeces of
four healthy donors
Isolation medium MIC No. of Bacterial species MIC50 MIC90
(µg/ml) strains (no. of strains) (µg/ml) (µg/ml)
Brain-heart 0.5 4 E. faecalis (3) 1 64
infusion agar E. faecium (1)
1 18 E. faecalis
2 3 E. coli
8 1 E. coli
16 2 E. coli
64 9 E. faecalis
128 2 E. durans (1)
E. faecalis (1)
>128 2 E. faecalis
McConkey 1 2 E. coli 8 8
2 2 E. coli
8 6 E. coli
Enterococcus 1 8 E. faecalis (7) 1 64
Lc lactis lactis (1)
64 3 E. faecalis
Brain-heart <0.125 2 Clostridium spp. (1) 1 32
infusion agar Bacteroides spp. (1)
0.25 3 Propionobacterium acnes (1)
Bifidobacterium breve (1)
Clostridium spp.
0.5 7 Gram-negative bacilli (1)
Mobiluncus spp. (3)
Gram-positive bacilli (1)
Bifidobacterium breve (2)
1 6 Bifidobacterium spp. (4)
Bifidobacterium infantis (1)
Bifidobacterium longum (1)
8 1 Gram-positive bacilli (1)
16 2 Bacteroides spp. (1)
Clostridium spp. (1)
32 2 Bacteroides caccae (1)
Bacteroides vulgatus (1)
64 2 Bacteroides distasonis (1)
Bacteroides uniformis (1)
128 1 Bacteroides vulgatus (1)
Table 1 (continued)
Isolation medium MIC No. of Bacterial species MIC50 MIC90
(µg/ml) strains (no. of strains) (µg/ml) (µg/ml)
Schaedler <0.125 2 Clostridium spp. (1) 1 64
Bacteroides merdae (1)
0.25 1 Bacteroides spp.
0.5 5 Gram-positive bacilli (1)
Bifidobacterium infantis (1)
Bifidobacterium breve (1)
Bacteroides spp. (1)
Actinomyces israeli (1)
1 10 Mobiluncus spp. (4)
Bifidobacterium spp. (3)
Bifidobacterium longum (1)
Clostridium ramosum (1)
Peptostreptococcus
asaccharolyticus (1)
16 3 Gram-positive bacilli (1)
Bacilli (2)
32 4 Gram-positive bacilli (1)
Bacteroides spp. (1)
Bacteroides uniformis/ovatus (2)
64 5 Bacteroides ovatus (1)
Bacteroides vulgatus (3)
Bacteroides spp. (1)
content (Van Marwyck, 1958) and that the bacterial content of the
chemostats simulates that of the colon on a daily basis. Daily samples
were taken before treatment (days 17-24) and during treatment (days
25-33).
In a separate set of experiments, chemostat faecal cultures were
used to evaluate the emergence of resistant strains and loss of
colonization resistance to a Clostridium difficile strain. The
methods were validated for short-chain fatty acids, enzymes, bile
acids (primary and secondary analytes), protein concentration,
cellular fatty acids, and sulfate reduction. Additional studies were
performed to validate the procedures for maintaining the chemostat
cultures.
(i) Microfloral ecology
In the studies described below, the chemostats were inoculated
with the faecal pool and allowed to reach steady-state, which required
about two weeks. Samples were taken daily before (days 17-24) and
during treatment (days 25-33).
Cellular fatty acid profile: Cellular fatty acid profiles were
used to determine whether the bacterial cultures were changing and the
antibiotic was altering the bacteria. Each sample was centrifuged and
washed in buffer, and the bacterial pellet was collected. The cellular
fatty acids were extracted, derivatized, and identified and quantified
by gas chromatography. The profiles were compared statistically with
Microbiological Identification Inc. (Newark, DE) software developed to
identify unknown strains of bacteria. The unit of relatedness of two
cellular fatty acids profiles is the Euclidian distance (ED). Similar
profiles have a low ED and different profiles have a high ED.
Statistically significantly identical profiles (e.g. the same sample
run twice) are related by < 2 ED; different strains of the same
subspecies are related by an ED of 2-6; different subspecies of the
same species are related by an ED of 6-10; and different species from
a single genus are related by an ED of 10-25. These values were
established by analysing thousands of previously characterized
anaerobic bacteria from culture collections.
In the chemostats, the flora on day 1 of inoculation resembles
that of the faeces. After inoculation, the flora changes until it
reaches steady-state, i.e. an unchanging bacterial community. Higher
EDs indicate increasing differences between the successive daily
profiles of the chemostat and the faecal flora. As the flora reaches a
steady-state, the ED is lower. For example, the results on incubation
day 7 differ from those on day 6 by an ED of 2, indicating that the
flora is almost identical on those two days and that the chemostat is
reaching steady-state. The steady-state flora and faeces had different
cellular fatty acid profiles, however, indicating a difference in
either the number of bacterial species or the cellular fatty acid
composition of the flora.
After establishment of the steady-state about two weeks after
inoculation, tetracycline was added to the chemostats at 0, 0.15, 1.5,
or 15 mg/L, and the range of relatedness values generated for each
successive daily sample was compared with the total range of values.
An effect of the drug would be reflected as an increase in the ED
value with increasing dose of tetracycline in the chemostat; however,
as seen in Table 2, the results showed no difference in ED at the four
doses tested when daily samples were analysed before and during
treatment. This suggests that tetracycline does not alter the overall
composition of cellular fatty acids in the chemostats.
Short-chain fatty acids: The three main short-chain fatty acids
produced by the intestinal microflora as a product of bacterial
metabolism are acetic, propionic, and butyric acids. These fatty acids
are absorbed from the colon. Butyrate is the main energy source for
colonic enterocytes. Propionate is transported to the liver and
metabolized and reduces serum cholesterol. Acetate enters the
peripheral circulation, where it is metabolized. Since the objective
of this study was to model the environment of the intestinal flora,
the net concentrations of short-chain fatty acids were used as a
measure of bacterial metabolism.
Because of the complexity of the intestinal microflora and lack
of knowledge about bacterial fatty acids in vivo, there are no fatty
acid markers for particular groups of bacteria. This is however a
sensitive assay commonly used for analysing the total intestinal
microflora. Short-chain fatty acids were extracted from chemostat
samples, derivatized, and analysed by gas chromatography. This method
provides information only on changes in the fatty acid profiles due to
treatment.
The short-chain fatty acid content of the chemostats reached
steady-state at the beginning of the third week. Samples were taken
daily before and during treatment. The total short-chain fatty acid
concentrations of the chemostats containing the drug were similar to
those of the control chemostat. In addition, tetracycline had no
effect on the concentrations of individual fatty acids or on the
combined concentration of the minor short-chain fatty acid components.
The concentrations were almost uniform between the chemostats,
constant within chemostats at steady-state, and unaffected by the
addition of tetracycline. The only exception was the control
chemostat, in which the concentration of the combined minor
short-chain fatty acids rose at the expense of the propionate. Since
fatty acids are common indicators of changes in the metabolism of
bacteria in the intestinal microflora, these results show that
tetracycline does not alter the metabolic activity of the flora or
that the flora can compensate for the effect of the drug at the
concentrations tested.
Table 2. Relatedness of cellular fatty acid profiles of faecal
flora after treatment with tetracycline in a continuous flow
chemostat system
Dose Relatenessa
No drug (0 ADI) 11.5
0.15 mg/L (1 ADI) 17.1
1.5 mg/L (10 ADI) 11.1
15 mg/L (100 ADI) 13.3
a Euclidian distance
Bile acids: The concentration of bile acids produced during
lipid metabolism is related to fat intake and is a high-risk factor
for metastatic bowel disease. High concentrations of bile acids
produced in response to high fat intake stimulate the growth of
certain bacterial species, such as the Bacteroides. While these
bacteria are dying and before they are excreted in faeces, their
enzymes are liberated. Some of these enzymes can metabolize primary
bile acids into secondary genotoxic metabolites. When the fat intake
is high and fibre intake low, these genotoxic secondary metabolites
concentrate in the intestinal lumen and remain in the colon for a
longer time, allowing high concentrations of genotoxic metabolites to
come into contact with the intestinal cells for prolonged periods.
Primary (mammalian) and secondary (bacterial metabolites) bile acids
can be measured in two ways: as the levels of individual and total
analytes and as the percentage of the total that is secondary bile
acids. The latter was measured in this study, since the concentration
of primary bile acids in the medium was fixed. Two primary bile acids,
cholic and chenodeoxycholic acids, were added to the medium.
The results did not show a clear effect of the drug. For example,
the percent molar ratio of chenodeoxycholic acid increased at 1.5 mg/L
and decreased at 0, 0.15, and 15 mg/L during the steady-state period,
for no apparent reason. The percent of secondary bile acids was
similar and uniform at all concentrations, except in the control
chemostat where the concentrations continued to rise steeply during
the steady-state phase. In general, the dose of tetracycline had no
effect on the percent of secondary bile acids, but the background
(control) data were very variable.
Neutral sterols: The main difference between bile acids and
primary neutral animal sterols is that the latter lack the carboxylic
acid group of bile acids; however, their biological properties are
similar. Primary neutral sterols are converted to secondary sterols by
bacterial metabolism. The commonest animal neutral sterol is
cholesterol, and its concentrations in the culture medium can be
adjusted. In the intestine, cholesterol is converted to coprostanol
via an intermediate metabolite, coprostanone. Eubacterium spp. are
important cholesterol converters in the human intestine. The parameter
measured in the study was the percentage of secondary neutral sterols
present in the medium before and during treatment, indicative of
bacterial metabolism. Tetracycline had no noticeable effect on the
conversion of cholesterol to coprostanol.
(ii) Bacterial enzymes
Bacterial enzymes were analysed in chemostat sample effluent with
chromogenic substrates. The results were expressed as activity per
milligram of protein.
Sulfate reduction: The concentration of sulfate in the human
colon ranges from 1 to 16 mmol/d, depending on the diet. The daily
flow of inorganic sulfate in the chemostat was about 1.5 mmol, organic
sulfate being present at some proportion. The chemostat medium is thus
a good approximation of the human sulfate colon content. The parameter
measured in this study was the content of sulfide, a metabolite of
sulfate generated by bacterial metabolism. Sulfate reduction was
uniform at steady-state and remained unchanged throughout treatment in
all chemostats. Thus, no effects of tetracycline were seen.
Azoreductase: Anaerobic colonic bacteria that use azo compounds
as an energy source reduce azo dyes. No clear drug-related pattern was
observed on this parameter; however, although the short-chain fatty
acid concentrations indicated that the chemostats had reached
steady-state, the azoreductase concentrations of the treated
chemostats continued to decrease in the fourth week of the study.
Nitroreductase: Nitroreductase, a bacterial oxygen-labile
enzyme, was also measured anaerobically. The results were difficult to
interpret. Two chemostats, containing 0.15 and 15 mg/L, showed steady
declines in nitroreductase activity during the presumed steady-state
phase; however, the control chemostat and that containing 1.5 mg/L had
constant activity at steady-state. After the addition of 15 mg/L
tetracycline, the decline was reversed. No conclusions were drawn from
this study because the effect seen at the high dose was not seen at
the lower doses and the data were highly variable for all of the other
chemostats.
beta-Glucosidase: beta-Glucosidase is produced by several
colonic bacteria, including Bacteroides, Streptococcus, and
Lactobacillus spp.; it hydrolyses many sugar conjugates. When this
enzyme is present at high concentrations in the colon, the
glycone-linked compounds are deconjugated and enter the enterohepatic
circulation. Since many conjugated compounds, including bile acids,
are potentially genotoxic, high concentrations of glucosidase activity
have been linked to an increased risk for colon cancer. High
concentrations of glucosidase are present in people who eat high-fat,
low-fibre diets. In this study, the four chemostats had different
levels of beta-glucosidase activity at steady-state; the levels varied
during treatment but showed no trend after the addition of
tetracycline.
beta-Glucuronidase: Most of the beta-glucuronidase activity in
the colon is due to Bacteroides spp., although E. coli and other
species also produce this enzyme. When the glucuronide conjugate is
hydrolysed by bacterial metabolism, it enters the enterohepatic
circulation. The activity of beta-glucuronidase is influenced by diet,
as high-fat diets induce production of this enzyme. In this study, the
activity of beta-glucuronidase was still falling in the control
chemostat and that containing 1.5 mg/L and rising in those with 0.15
and 15 mg/L even at steady-state. Addition of tetracycline did not
change the trends.
These studies on several bacterial enzymes failed to show a clear
effect of tetracycline. While some parameters used to monitor
bacterial ecosystems indicated that a steady-state was achieved, the
enzyme concentrations were still variable. This inherent variability
may limit the usefulness of enzymatic activity for determining the
effect of antibiotics in vitro.
(iii) Bacterial counts
Techniques developed at the Virginia Polytechnic Institute and
State University (VPI) Anaerobe Laboratory and those described in the
Wadsworth Anaerobic Bacteriology Manual were used to count anaerobic
bacteria. In addition, coliforms and enterococci were cultured
aerobically, and the ratios of coliforms or enterococci to total
anaerobe counts were reported. Bacteroides fragilis, fusobacteria,
bifidobacteria, clostridial spores, eubacteria, veillonella,
peptostreptococci, peptococci, megasphaera, E. coli, and enterococci
were also counted. Although bacterial counts are traditionally used to
assess perturbations in intestinal microflora, total bacterial counts
lack specificity for different treatment groups, and the counts for
matched samples vary widely. Although total anaerobes were more
numerous in the chemostat after addition of the highest dose of
tetracycline, it had no major effects on bacterial counts.
2.3.1.3 Effect of tetracyclines on colonization resistance
The effects of tetracyclines on resistance were studied in
another series of studies conducted by Techlab Inc. (1997).
Steady-state was reached about two weeks after chemostats were
inoculated with faeces. After the pretreatment period (days 17-24),
tetracycline at doses of 0, 0.15, 1.5, or 15 mg/L was added on days
25-33. On days 33, 34, and 35, each chemostat was inoculated with
107 viable cells of Clostridium difficile VPI 10463 in order to
study the ability of the flora to prevent colonization with a foreign
microorganism. Samples were drawn between days 34 and 42. Control
samples were taken before and during tetracycline treatment to obtain
the background count and toxin titre. Samples were cultured for
C. difficile in selective medium [D-cycloserine, cefotoxin, and
fructose agar (CCFA)] with and without prior washing with ethanol for
additional selection of spores. The CCFA medium allows growth of other
Clostridium species and of at least one Enterococcus sp. if
washing with ethanol is not performed. The concentrations of
C. difficile toxin A were also measured. A positive control
chemostat was inoculated with C. difficile VPI 10463 but no faeces
in order to show that C. difficile can colonize the chemostat. The
counts in this chemostat decreased during the first 72 h, but once
C. difficile had adapted to the chemostat conditions (about day 6
after inoculation), the counts rose to 105-106 microorganism per
ml.
The chemostats were not colonized by C. difficile, unlike the
positive control chemostat, and toxin A production was not detected
after the initial period of inoculation. These results could not be
confirmed by culture of C. difficile on CCFA because in chemostats
inoculated with faeces the bacterial count on CCFA medium in the
absence of C. difficile was frequently as high or even higher than
the count in the control chemostat with no faeces and inoculated with
C. difficile. The species of bacteria that constituted this
background were not identified.
In summary, C. difficile colonized a positive control chemostat
(with no faeces added) lacking resistance to colonization. Addition of
tetracycline at doses of 0.15, 1.5, or 15 mg/L to chemostats
inoculated with faeces did not reduce the colonization resistance of
the steady-state flora to a level that would allow C. difficile to
colonize.
2.3.1.4 Selection of bacterial strains resistant to tetracycline
The numbers of tetracycline-resistant strains of enterococci,
E. coli, and Bacteroides fragilis were compared in control
chemostats and in chemostats treated with 0.15, 1.5, or 15 mg/L
tetracycline. First, the background resistance to tetracycline was
determined in the faecal pool used to inoculate the chemostats. The
percentages of isolated strains resistant to tetracycline are shown in
Table 3.
Table 3. Background resistance of faecal bacteria to tetracycline
Tetracycline Percent resistance
E. coli Enterococci
4 3 ND
8 3 91
16 3 92
32 ND 91
64 ND 91
ND, not determined
The increase in the number of resistant strains of E. coli was
measured against a background of 3% at 8 µg/ml. E. coli from
McConkey agar plates containing 20-300 colonies were plated in
replicate onto agar containing 4, 8, or 16 µg/ml tetracycline. After
16 and 24 h of incubation, the counts on the master plates were
compared with those on the plates with tetracycline. The highest dose
of tetracycline increased the percent of resistant E. coli from
< 20 to > 50% within 24 h, rising to > 60% after a second day of
exposure. Despite continued treatment, the percents of resistant
E.coli then began to fall, and by day 6 after treatment it was about
35%. In the control chemostat (no tetracycline), the percent of
resistant strains never exceeded 5%. The chemostats with 0.15 and 1.5
mg/L gave variable results. No effect was seen on the resistance of
enterococci or B. fragilis, probably because of the high level of
resistance (60œ100%) of these microorganisms. Furthermore, the
resistance of these organisms was already very high at the beginning
of the study (91% of the enterococci in the faecal pool used to
inoculate the chemostats were resistant to tetracycline), so that the
addition of tetracycline did not have any measurable effect. In the
chemostats, the lowest level of tetracycline resistance in
B. fragilis strains was > 50%, and most of the levels were close to
90%.
2.3.2 Animal models
In studies of germ-free mice associated with human flora,
treatment in drinking-water with 0.5 µg/ml chlortetracycline increased
the number of resistant E. coli. Although the dose was similar to
the residues allowed in food, the contribution of the
chlortetracycline residues to resistance is probably low, because it
has been shown that the daily increase in the number of resistant
enterobacteria after ingestion of food can be 1 000 000 (Corpet, 1987,
1988). In a study in which germ-free mice were associated with two
isogenic strains of E. coli, one of which carried an R plasmid, the
mice were given tetracycline in drinking-water for 10œ15 days. The
minimum selecting concentration of tetracycline was 6.5 µg/ml, which
resulted in a faecal concentration of 0.5 µg/g tetra-cycline -
one-half of the MIC for the plasmid-free strain. This concentration
did not eliminate the susceptible strains from the gut; however, this
model did not include the dominant anaerobic flora that is responsible
for the barrier effect (Corpet et al., 1989; Corpet, 1993). In a
study in which germ-free mice were associated with faecal microflora
from piglets and treated with 20 µg/ml chlortetracycline in the
drinking-water, the occurrence of lactose-fermenting bacteria
resistant to chlortetracycline increased (Corpet, 1984). Although the
effect of chlortetracycline was studied in piglet microflora, the
concentration of the drug in the water was much higher than the amount
of residues allowed in food. These studies suggest that the variations
seen in human faecal flora after exposure to low concentrations of
tetracyclines in food might be similar to the variation that occurs
naturally.
2.4 Studies in humans
The emergence of resistant E. coli strains in humans after
therapeutic doses of tetracycline has been documented; however, the
level of resistant bacteria decreased with time after the last
treatment (Hirsh et al., 1973; Bartlett et al., 1975).
In early studies, some people given oxytetracycline at 10 mg/day
for six months showed an increased number of resistant coliforms and
yeasts; however, the effect was transitory, and resistant coliforms
were present even before treatment (Goldberg et al., 1961).
A dose of 100 mg/d tetracycline given for long-term treatment of
acne vulgaris altered the resistance patterns of the microflora,
increasing the percentage of people with transferrable R factor
bacteria and the number of multiresistant strains (Valtonen et al.,
1976). In volunteers given 0, 50, or 1000 mg/d of tetracycline for
four days, however, the shedding of E. coli from the intestine was
increased at the high dose but not at the low dose, indicating that 50
mg of tetracycline do not perturb the barrier effect of the human
flora (Hirsh et al., 1974).
A dose-related effect of oxytetracycline was seen on intestinal
microflora in people given 2, 20, or 2000 mg/d. The effects ranged
from drastic disturbance of the microflora (elimination of susceptible
anaerobes and more resistant Enterobacteriaceae with 2 g/day) to a
tendency to a decrease in the number of susceptible Enterobacteriaceae
and an increase in the number of resistant strains at 20 mg/d. A dose
of 2 mg/d therefore appears to have no effect on the selection of
resistant Enterobacteriaceae (Tancrede & Barakat, 1987).
Healthy humans given oxytetracycline at 1 g/d orally for five
days showed a decreased faecal concentration of saponifiable
conjugates of bile acids, which are probably formed by metabolic
action of the intestinal microflora. The drug also reduced bacterial
transformation of cholesterol to coprostanol and coprostanone,
decreased the amount of faecal sterified neutral sterols, and
increased excretion of faecal conjugated oestrogens (Korpela et al.,
1984, 1986; Hamalainen et al., 1987). The same dose of tetracycline
has been shown to increase the number of tetracycline-resistant
strains of E. coli (Hirsh et al., 1973; Bartlett et al., 1975);
however, the minimum effective dose of tetracyclines, 1 g/d, has been
shown to have a minimal effect on the total population of aerobic or
anaerobic bacteria after 8-10 days of oral administration. When given
in divided doses, this dose of tetracycline also has infrequent, mild
side-effects (nausea, diarrhoea, and vomiting), which disappear when
treatment is discontinued (Kunin & Finland, 1961; Bartlett et al.,
1975).
3. COMMENTS
The studies used originally to establish an ADI for
oxytetracycline, tetracycline, and chlortetracycline showed that these
drugs have a low degree of toxicity, and additional toxicological
studies have confirmed that conclusion.
Because the tetracyclines are poorly absorbed from the
gastrointestinal tract, high concentrations are readily achieved in
the intestine, perturbing the intestinal microflora within 48 h of
daily treatment. Experience with tetracycline in human medicine
indicates that therapeutic levels of tetracyclines can perturb the
intestinal microflora by inducing resistant strains, altering the
metabolic activity of the microflora and the colonization resistance
properties of the flora (barrier effect), which allows overgrowth of
pathogenic, opportunistic, or resistant microorganisms, and altering
the ecological balance of the flora with no identified deleterious
effect.
Studies in humans have shown that 1-2 g/d of tetracyclines have
adverse effects on the intestinal microflora. Resistant strains
emerged in one study at doses of 100 mg per person per day of
tetracycline and 10 mg per person per day of oxytetracycline, but
resistant organisms existed before treatment and the resistance was
transitory. Another microbiological study in humans showed that 50 mg
tetracycline did not alter the shedding of E. coli from the
intestine, indicating that the barrier effect was not perturbed at
this dose. In the study used previously by the Committee to establish
the ADI for tetracyclines, a therapeutic dose of oxytetracycline
(2 g/d for seven days) perturbed the balance of the intestinal flora,
with an increased prevalence of resistant Enterobacteriaceae and yeast
colonization. A dose of 20 mg/d had a marginal ecological impact on
the flora by suppressing some susceptible anaerobes, but the dose of
2 mg/d did not result in the presence of resistant Enterobacteriaceae
in the faeces. The thirty-sixth Committee applied a 10-fold safety
factor to the 2 mg/d dose to establish an ADI of 0-0.003 mg/kg bw.
Since the previous evaluation, more data from microbiological studies
on the effects of tetracyclines on human gastrointestinal bacterial
flora have been obtained.
The results obtained in a continuous bacterial culture system
(chemostat) dosed to provide the equivalent of 0.025, 0.25, or 2.5
mg/kg bw per day of tetracycline showed that a dose of 2.5 mg/kg bw
per day slightly increased the number of resistant E. coli strains,
which increased from < 20% to > 50% within 24 h of exposure and to
> 60% after 48 h of exposure; however, the percentage fell to about
35% by day 6 of exposure, despite continued treatment. In the control
chemostat, which contained no drug, the percentage of resistant
bacteria never exceeded 5%. No effects were seen in chemostats dosed
with tetracycline at the equivalent of 0.025 or 0.25 mg/kg bw per day.
None of the other microbiological end-points analysed in this study
changed with any concentration of tetracycline tested.
4. EVALUATION
The results are in agreement with findings reported in the
literature on the effects of tetracycline on human intestinal
microflora. Most report a very low impact of tetracycline on colonic
flora, selection of resistant bacterial strains being the most
sensitive end-point. On the basis of the new information on the effect
of tetracycline on human intestinal microflora, the Committee
concluded that no safety factor was needed, because the selection of
resistant Enterobacteriaceae is a very sensitive end-point for
evaluating the microbio-logical effect of tetracyclines on human
intestinal microflora and because individuals show little variation
with respect to this effect. On the basis of the NOEL of 2 mg/d
(equivalent to 33 µg/kg bw per day) in the study in humans, the
Committee established an ADI of 0-30 µg/kg bw for the tetracyclines,
alone or in combination. The ADI was rounded to one significant
figure, as is usual practice.
5. REFERENCES
Bartlett, J.G., Bustetter, L.A., Gorbach, S.L. & Onderdonk, A.B.
(1975) Comparative effect of tetracycline and doxycycline on the
occurrence of resistant Escherichia coli in the fecal flora.
Antimicrob. Agents Chemother., 7, 55-57.
Brady, M.S. & Katz, S.E. (1988) Method to determine effect of
antibiotics at residue levels on R-factor transfer. J. Assoc.
Anal. Chem., 71, 299-301.
Brady, M.S. & Katz, S.E. (1992) In vitro effect of multiple
antibiotic/antimicrobial residues on the selection of resistance in
bacteria. J. Assoc. Anal. Chem. Int., 75, 738-742.
Brady, M.S., Strobel, R.J. & Katz, S.E. (1988) In vitro analytical
system for determining the ability of antibiotics at residue levels to
select for resistant bacteria. J. Assoc. Anal. Chem., 71, 295-298.
Brady, M.S., White, N. & Katz, S.E. (1993) Resistance development
potential of antimicrobial residue levels designated as 'safe levels'.
J. Food Protect., 56, 229-233.
Brown, S.A. (1988) Treatment of gram-negative infections. Vet.
Clin. N. Am. Small Anim. Pract., 18, 1141-1165.
Chopra, I., Hawkey, P.M., & Hinton, M. (1992) Tetracyclines, molecular
and clinical aspects. J. Antimicrob. Chemother., 29, 245-277.
Centre National d'Etudes Vétérinaires et Alimentaire (1997) Use of
human flora-associated rodents to study the effect of low doses of
tetracyclines and neomycin: Pilot study. Unpublished second quarterly
report. Submitted to WHO by Department of Health and Human Services/US
Food and Drug Administration, Rockville, Maryland, USA.
Corpet, D.E. (1984) The effect of bambermycin, carbadox,
chlortetracycline and olaquindox on antibiotic resistance in
intestinal coliforms: A new animal model. Ann. Microbiol. (Inst.
Pasteur), 135A, 329-339.
Corpet, D.E. (1987) Antibiotic residues and drug resistance in human
intestinal flora. Antimicrob. Agents Chemother., 31, 587-593.
Corpet, D.E. (1988) Antibiotic resistance from food. New Engl. J.
Med., 318, 1206-1207.
Corpet, D.E. (1993) An evaluation of methods to assess the effect of
antimicrobial residues on the human gut flora. Vet. Microbiol., 35,
199-212.
Corpet, D.E. & Brugère, H.B. (1996) Antimicrobial drug residues in
foods: Evaluation of the no effect level on human microflora.
Recent Res. Devel. Antimicrob. Agents Chemother., 1, 113-123.
Corpet, D.E., Lumeau, S. & Corpet, F. (1989) Minimum antibiotic levels
for selecting a resistance plasmid in a gnotobiotic animal model.
Antimicrob. Agents Chemother., 33, 535-540.
Egger, R. & Lebek, G. (1985) Selection of R-plasmid harbouring
E. coli by subinhibitory concentrations of antibiotics. Zbl.
Bakt. Hyg. A, 260, 361-368.
Goldberg, H.S., Goodman, R.N., Logue, J.T. & Handler, F.P. (1961)
Long-term, low-level antibiotics and the emergence of antibiotic-
resistant bacteria in human volunteers. Antimicrob. Agents
Chemother., 80-88.
Gorbach, S.L. (1993) Perturbation of intestinal microflora. Vet.
Hum. Toxicol., 35 (Suppl. 1), 15-23.
Hamalainen, E., Korpela, J.T. & Adlercreutz, H. (1987) Effect of
oxytetracycline administration on intestinal metabolism of oestrogens
and on plasma sex hormones in healthy men. Gut, 28, 439-445.
Hirsh, D.C., Burton, G.C. & Blenden, D.C. (1973) Effect of oral
tetracycline on the occurrence of tetracycline-resistant strains of
Escherichia coli in the intestinal tract of humans. Antimicrob.
Agents Chemother., 4, 69-71.
Hirsh, D.C., Burton, G.C., Blenden, D.C. & Tsutakawa, R. (1974) The
effect of tetracycline upon establishment of Escherichia coli of
bovine origin in the enteric tract of man. J. Appl. Bacteriol., 37,
327-333.
Korpela, J.T., Hamalainen, E. & Adlercreutz, H. (1984) Effect of
oxytetracycline on bacterial intestinal metabolism of neutral sterols
and on serum lipids. Scand. J. Gastroenterol., 19, 401-404.
Korpela, J.T., Fotsis, T. & Adlercreutz, H. (1986) Multicomponent
analysis of bile acids in faeces by anion exchange and capillary
column gasœliquid chromatography: Application in oxytetracycline
treated subjects. J. Steroid. Biochem., 25, 277-284.
Kunin, C.M. & Finland, M. (1961) Clinical pharmacology of the
tetracycline antibiotics. Clin. Pharmacol. Ther., 2, 51-61.
Lindenbaum, J., Rund, D.G., Butler, V.P., Tse-Eng, D., & Saha, J.R.
(1981) Inactivation of digoxin by the gut flora: Reversal by
antibiotic therapy. New Engl. J. Med., 305, 789-793.
Nord, C.E., Kager, L. & Heimdahl, A. (1984) Impact of antimicrobial
agents on the gastrointestinal microflora and the risk of infections.
Am. J. Med., 76, 99-106.
Ramos, K.S., Chacon, E. & Acosta, D., Jr (1996) Toxic responses of the
heart and vascular system. In: Casarett & Doull's Toxicology: The
Basic Science of Poisons, 5th Ed., New York, McGraw-Hill, pp.
487-527.
Richez, P. (1994) Antibacterial activity of tetracycline
oxytetracylcine against human gut flora. Study No. SVL 029.
Unpublished report from Datavet Veterinary Drug Consultants. Submitted
to WHO by Intervet International, Boxmeer, Netherlands
Roberts, M.C. (1996) Tetracycline resistance determinant: Mechanisms
of action, regulation of expression, genetic mobility, and
distribution. FEMS Microbiol. Rev., 19, 1-24.
Sande, M.A. & Mandell, G.L. (1990) Tetracyclines, chloramphenicol,
erythromycin, and miscellaneous antibacterial agents. In: Goodman
and Gilman's The Pharmacological Basis of Therapeutics, 8th Ed., New
York, Pergamon Press, pp. 1117-1125.
Tancrede, C. & Barakat, R. (1987) Ecological impact of low doses of
oxytetracycline on human intestinal microflora. J. Vet. Med., 42,
35-39.
Techlab, Inc. (1997) Implementation of an in vitro system to study the
effect of low doses of antimicrobials on the human intestinal flora.
Unpublished 6th, 7th and 8th annual reports. Submitted to WHO by
Department of Health and Human Services/US Food and Drug
Adminstration, Rockville, Maryland, USA.
Valtonen, M.V., Valtonen, V.V., Salo, O.P. & Makela, P.H. (1976) The
effect of long term tetracycline treatment for acne vulgaris on the
occurrence of R factors in the intestinal flora of man. Br. J.
Dermatol., 95, 311-316.
Van Marwyck, C. (1958) The excretion of tetracycline via the feces.
Munch. Med. Wochenschr., 100, 1-5 (in German).
See Also:
Toxicological Abbreviations