The effect of bambermycin, carbadox, chlortetracycline and olaquindox on antibiotic resistance in intestinal coliforms: a new animal model

Groups of germ-free mice kept in isolators and associated with faecal microflora from piglets were continuously given either water or a solution of one of the following: chlortetracycline (20 micrograms/ml), carbadox (50 micrograms/ml), olaquindox (50 micrograms/ml), bambermycin (flavomycin) (5 micrograms/ml) or mixtures of these drugs. The proportions of lactose-fermenting bacteria in their faeces which were resistant to chlortetracycline, carbadox or olaquindox were measured by a comparative plate-counting procedure. Compared to occurrence in control mice, the occurrence of antimicrobial drug-resistant bacteria was higher in mice receiving chlortetracycline (P less than 0.001) and lower in mice receiving bambermycins (P less than 0.005). In contrast, olaquindox and carbadox did not change the proportion of resistant coliforms in mice faeces. A control experiment was conducted with five groups of germ-free mice given the same flora and kept without drugs in separate isolators. No difference in the occurrence of resistant coliforms could be found between these groups. The germ-free mouse associated with faecal microflora from a conventional animal seems to be a suitable model for determining in vivo the effect of low doses of antimicrobial drugs on drug resistance in lactose-fermenting enteric flora

An evaluation of methods to assess the effect of antimicrobial residues on the human gut flora

1. Barrier effect. Relevant models should include an anaerobic dominant flora that antagonizes minor bacterial populations such as drug resistant E. coli. 2. Anaerobes vs. aerobes. Aerobe counts are more precise and much less time consuming than anaerobe counts. Minor populations of drug resistant aerobes are sensitive markers of the ecosystem balance, and are directly relevant to the potential risk of antimicrobial residues. 3. MIC vs. plate counts. The determination of minimum inhibitory concentrations ( MIC ) of selected clones is time consuming, does not detect subdominant resistance (less than 1 %), and the MIC shift is difficult to test statistically. In contrast, direct counts of bacteria on drug supplemented media allows a rapid measure of minor resistant populations. 4. Statistics: Most published designs do not include adequate statistical evaluation. This is critical for trials made in conventional humans and animals, where data are highly variable. 5. Human trials: The lowest concentration of antibiotic tested in human volunteers (2mg oxytetracycline /d for 7d in 6 subjects) significantly increased the proportion of resistant fecal enterobacteria (P=0.05). However, the huge day-to-day and inter-individual variations of human floras make this evidence rather weak. 6. Gnotobiotic mice inoculated with human flora are living isolated models in which the effect of any antimicrobial on the human gut flora can be tested. This in vivo model does include the barrier effect of dominant anaerobes. Inter-individual and day-to-day variations of bacterial populations are lower in those mice than in humans. 7. Most resistant enterobacteria in the human gut of untreated people come from bacterial contamination of raw foods. The relative contribution of residues in selecting antibiotic resistance seems to be low when compared to bacterial contamination

Red meat and colon cancer : should we become vegetarians, or can we make meat safer ?

The effect of meat consumption on cancer risk is a controversial issue. However, recent meta-analyses show that high consumers of cured meats and red meat are at increased risk of colorectal cancer. This increase is significant but modest (20-30%). Current WCRF-AICR recommendations are to eat no more than 500g per week of red meat, and to avoid processed meat. Moreover, our studies show that beef meat and cured pork meat promote colon carcinogenesis in rats. The major promoter in meat is heme iron, via N-nitrosation or fat peroxidation. Dietary additives can suppress the toxic effects of heme iron. For instance, promotion of colon carcinogenesis in rats by cooked, nitrite-treated and oxidized high-heme cured meat was suppressed by dietary calcium and by α-tocopherol, and a study in volunteers supported these protective effects in humans. These additives, and others still under study, could provide an acceptable way to prevent colorectal cancer

Antibiotic resistance from food

Antimicrobial resistant Escherichia coli are found in most fecal samples from the normal population. The present study tested the hypothesis that antibiotic-resistant bacteria come from contaminated food. Six healthy volunteers ate a sterile diet for 3 weeks after a control period. The fecal incidence of resistance to ampicillin, tetracycline, and streptomycin in lactose-fermenting enteric bacilli was determined daily. During the control period, the populations of fecal resistant lac+ enteric bacilli varied with time, periodically reaching a high level of 108 per gram. After the start of the sterile diet, the fecal concentration of resistant bacteria dropped. Three days later, no resistant strain could be detected in the feces of three volunteers, whereas in those of the other three, some could be detected sporadically. The sterile diet reduced the number of resistant bacilli in all volunteers (p<0.001). Thus, most fecal resistant lac+ enteric bacilli come from contaminated food: transient strains enter the intestines with food and are excreted in feces

Energy balance and cancers

Energy balance results from the exact equilibrium between caloric intake and caloric expenditure. A caloric intake larger than caloric expenditure results in overweight, even obesity, but other determinants, like hormonal dysfunction and/or genetic traits may play a part in obesity syndrome. Obesity, and even overweight, have been recognized as risk factors for the development of cancers. Human epidemiological studies, which have tended to establish the nature of the relationship between energy balance and cancer, are summarized first, with the influence of the various factors which act both on obesity and on cancer risk. Among these factors are the macronutrients responsible for the caloric intake, and some lifestyle factors (physical activity, drinking habits and tobacco use). Second, the animal studies help to distinguish between different relevant factors, and to understand some of the underlying mechanisms. However, the insulin-resistance syndrome, which appears to underlie the relationship between obesity and hormone-dependent cancers, and possibly colon cancer, is only relevant to human physiology because hormonal alterations are part of it. Prevention of hyperinsulinemia, insulin resistance and the accompanying visceral obesity appears to be a major public health task for the prevention of cancers

Antibiotic residues and R-plasmid selection: are in vitro methods good models?

Three clones of E. coli, one of which was harbouring a tetracycline resistance plasmid were inoculated together into the stomach of axenic mice. Without antibiotic selective pressure, the R-Plasmid bearing strain became dominant in the faeces of mice, while the R-plasmid free strain was eliminated. When the R-plasmid bearing strain was given to mice 4 days after the inoculation with the R-plasmid free strain, it was repressed and remained at the stable level of 10(4.5) organisms per g of faeces. But a rapid spread of the R-plasmid was observed, tetracycline resistant bacteria become dominant within one day, and replace the tetracycline sensitive E. coli. The tetracycline resistance plasmid did not disadvantage the mediating strain in the gut, even in the absence of antibiotic pressure. In contrast Lebek and Egger (1983), studying the same strains in vitro, found that in a chemostat the plasmid bearing strain was overgrown by the plasmid free strain. These results strongly suggest that in vitro interactions between E. coli strains cannot be directly extrapolated to in vivo conditions. For the determination of the no-effect level of antibiotic residue on the selection of R-factor in the gut, studies should be made in vivo

Point: From animal models to prevention of colon cancer. Systematic review of chemoprevention in min mice and choice of the model system.

The Apc(Min/+) mouse model and the azoxymethane (AOM) rat model are the main animal models used to study the effect of dietary agents on colorectal cancer. We reviewed recently the potency of chemopreventive agents in the AOM rat model (D. E. Corpet and S. Tache, Nutr. Cancer, 43: 1-21, 2002). Here we add the results of a systematic review of the effect of dietary and chemopreventive agents on the tumor yield in Min mice. The review is based on the results of 179 studies from 71 articles and is displayed also on the internet http://corpet.net/min.(2) We compared the efficacy of agents in the Min mouse model and the AOM rat model, and found that they were correlated (r = 0.66; P < 0.001), although some agents that afford strong protection in the AOM rat and the Min mouse small bowel increase the tumor yield in the large bowel of mutant mice. The agents included piroxicam, sulindac, celecoxib, difluoromethylornithine, and polyethylene glycol. The reason for this discrepancy is not known. We also compare the results of rodent studies with those of clinical intervention studies of polyp recurrence. We found that the effect of most of the agents tested was consistent across the animal and clinical models. Our point is thus: rodent models can provide guidance in the selection of prevention approaches to human colon cancer, in particular they suggest that polyethylene glycol, hesperidin, protease inhibitor, sphingomyelin, physical exercise, epidermal growth factor receptor kinase inhibitor, (+)-catechin, resveratrol, fish oil, curcumin, caffeate, and thiosulfonate are likely important preventive agents

Antagonistic Effect of Intestinal Bacteria from the Microflora of Holoxenic (Conventional) Piglets, Against Clostridium Perfringens in the Digestive Tract of Gnotoxenic Mice and Gnotoxenic Piglets

Antagonistic effect of piglet microflora against Clostridium perfringens was studied in germfree mice, to isolate bacterial strains responsible for this colonization resistance. The 1:100 dilution of the feces of a 2 day-old conventional piglet, given per os to germfree mice already harboring C. perfringens, led to the elimination of C. perfringens. From this piglet flora, 8 bacterial strains were selected, belonging to the genera Bacteroides, Clostridium, Eubacterium, Bifidobacterium, Lactobacillus and a strain belonging to the class of Mollicutes. When the 8 strains were given to germfree mice 3 days after C. perfringens inoculation, they led to rapid elimination of C. perfringens from feces. Sixteen other mixtures of 2 to 7 strains were similarly tested, but none was able to fully antagonize C. perfringens. When the 8 strains were given per os to germfree piglets after C. perfringens inoculation, they led to the rapid elimination of C. perfringens from pig feces, and to a quick recovery from diarrhea. This study led to the identification of a simplified fraction of gut microflora, able to exert a barrier effect against C. perfringens comparable to the entire flora of the piglet. This study suggests that gnotoxenic mice can be a suitable model for simplifying the flora responsible for a given effect in another host, animal or human

Mécanismes de la promotion de croissance des animaux par les additifs alimentaires antibiotiques [Mechanism of antimicrobial growth promoters used in animal feed]

Most feeds for broilers, pigs and veal calves, but 1/3 of feeds for beef cattle, are supplemented with an antimicrobial growth promoter. A European regulation list allows antimicrobial growth promoter, concentrations, animal species, and withdrawal periods (often null). Presently, avilamycin, flavomycin, lasalocid, monensin, and salinomycin are allowed. Avoparcin, bacitracin, carbadox, olaquindox, spiramycin, tylosin, and virginiamycin use was suspended by EU in 1997 and 98. Permitted doses are around 20 ppm (=20 g/t feed). Animals given antimicrobial growth promoter supplemented feeds grow faster and more uniformly than controls (average daily gain improved by 3 to 9%). They have a lower feed intake per weight gain (the feed conversion ratio is reduced by 3 to 12%), and release less nitrogen in the environment. The mechanism of growth promotion by antimicrobial growth promoters is still speculative. However, the growth promotion is likely due to a modification of the gut flora, since antimicrobial growth promoters do not promote the growth of germfree animals. Moreover, a modified antimicrobial growth promoter molecule promotes growth only as long as it retains antimicrobial potency. Most studies show no or little changes in the composition of gut flora during antimicrobial growth promoter supplementation. By contrast, antimicrobial growth promoters strongly suppress the bacterial catabolism of urea and amino-acids, both in vitro and in vivo. Antimicrobial growth promoters also decrease the fermentation of carbohydrates and the decomposition of bile salts. These changes increase the availability of nutrients and energy to the animal host (sparing effect), and decrease the concentration of toxic molecules like ammonia or amines in the gut, leading to a reduced turnover in the gut epithelium. In cattle, the rumen flora fermentations are oriented toward propionic acid production, instead of acetic acid, favouring the meat deposit, and less methane is produced. Because antimicrobial growth promoters show maximum efficacy in farms with poor hygiene, some authors argue that another beneficial effect of antimicrobial growth promoters lies in an improved control of pathologies (e.g., necrotic enteritis in poultry, swine dysentery in pigs)

Influence de faibles doses de chlortétracycline sur la résistance à la chlortétracycline de escherichia coli, dans le tube digestif de souris axéniques hébergeant des flores complexes d'enfant, de veau ou de porcelet

Afin de démontrer l'action de faibles doses d'antibiotiques sur le niveau de résistance des populations de Escherichia coli du tube digestif, des microflores fécales de veau, de porcelet et/ou de nouveau-né ont été inoculées à des souris axéniques. Celles-ci ont ensuite reçu de façon continue 20 µg de chlortétracycline/ml d'eau de boisson. Chez les animaux holoxéniques de départ, qui n'ont jamais reçu d'antibiotique, le nombre de E. coli résistants à la chlortétracycline est élevé, et égal environ au 1/l0e du nombre de bactéries sensibles. Sous l'influence de l'antibiotique le nombre de E. coli résistants a augmenté de 1,5 à 3 puissances de 10 chez nos trois modèles qui, maintenus en isolateur, ne pouvaient pas être contaminés par les bactéries extérieures. Malgré l'administration continue d'antibiotique le nombre de E. coli sensibles n'a pas été modifié, et il est resté toujours supérieur au nombre de résistants