Impact Of Tulathromycin On Human Colonic Microflora In Chemostat Models

As a novel macrolide antibiotic for animals, tulathromycin has lots of advantages such as lasting effect, long elimination half-life etc. Meanwhile, It may bring adverse affects to human health and food safty caused by residues in cattle and pigs edible tissue. At present, the microbiological safety evaluation on tulathromycin residues remains inadequate. The microbiological NOAELs/NOAECs by EMAE, FDA and APVMA were limited to the short-term antibiotic susceptibility tests in vitro, such as the MIC50, ignoring the long-term effects of the tulathromycin residues posed on the human intestinal bacteria, the amount, constitution of normal flora, metabolism, colonization resistance and resistance changes for instance. As a new veterinary drug, some potential side effects of tulathromycin have not been enough revealed. To address this concern, we conducted systematic studies to examine the effects of tulathromycin residues posed on human intestinal bacteria, so as to provide references for the rational microbiological ADI. Meanwhile, the conditioned pathogen may bring adverse affects to human health after long-term reaction with drug, a series of resistance and virulence experiments were conducted using Enterococcus faecalis, to determine its pathogenicity and harmfulness.1Impact of tulathromycin on two microbiological endpointsIn this study, we adopted a continuous culture chemostat model to study the effect of tulathromycin on human intestinal microflora. On the basis of the MIC50of the human intestinal bacteria to tulathromycin of official reports (EMEA,1.5μg/mL; APVMA,1μg/mL) and pre-experiment results, five concentration groups (0,0.1,1,10and100μg/mL) were choosed to exposed in the chemostat cultrue. Two important microbiological endpoints, colonization resistance and resistance changes, were investigated before and after treatment with drug. The colonization resistance consists of bacterial population, SCFA concentration, colonization of Salmonella Typhimurium. In low concentration group (0.1and1μg/mL), the colonization resistance and resistance were between the95%confidence, having no side effect with human intestinal microflora. While in medium-concentration group (10μg/mL), the Enterococci spp and Bacteroides fragilis number decreased significantly in18and16day, approximately1.6and6-fold as much as95%lower confidence limits. Just propionic acid of SCFA were slightly less than95%lower confidence limits, and without colonization of Salmonella Typhimurium, meanwhlie, the resistance rate trends of Enterococci spp and Bacteroides fragilis were slightly upwards, and all this show that the drug might pose a slight affects on human intestinal microflora in this concentration. In high concentration group (100μg/mL), the Escherichia coli, Bifidobacterium and Bacteroides fragilis number decreased approximately6,6and100-fold as much as95%lower confidence limits. The concentration of acetic acid, propanoic acid and butyric acid decreased approximately1.3,1.3and2.7-fold as much as95%lower confidence limits, meanwhile the number of Salmonella Typhimurium keep in105-106CFU/mL, and all this show that the drug might pose a significant effect on human intestinal microflora in this concentration, arousing intestinal microflora disorders.To sum up the results in this study and literature reported, we may get that the colonization resistance of human intestinal microflora is closely related to the number of Enterobacteriaceae.At the same time, the resistance rate of Escherichia coli, Enterococci spp and Bifidobacterium increased very significantly, so far as to80%for the later.the maximum no observable adverse effect concentration(NOAEC) to human intestinal flora of tulathromycin is1μg/mL, and the microbiological ADI is4.58μg/kg.bw/day (0.27mg/person/day), significantly lower than FDA, EMAE and APVMA microbiological ADI (50μg/kg.bw/day,10.97μg/kg.bw/day and5μg/kg.bw/day). The microbiological risk may be underestimated.2Impact of tulathromycin on resistance and virulence of Enterococcus faecalisEnterococcus faecalis were isolated and identificated before and after treatment with tulathromycin. Resistance phenotype, genetype and resistance transfer were determined in Enterococcus faecalis,. At the same time, five virulence factors, esp, cylA, gelE, ace and asal were determined to investigate the bacterial virulence, the relationship between virulence and resistance. The pathogenicity of Enterococcus faecalis harboring virulence factors were measured with SPF KM mouse.After treatment with tulathromycin, the MIC50of Enterococcus faecalis to tulathromycin increased from4.904to244.149, increased by49.8times, at the same time, it showed high levels of cross-resistance to macrolides, lincosamides and streptogramin B, typical MLSB resistance phenotype. Resistance is primarily mediated by the ermB gene (positive rate of88.46%), and the ermB gene is basically located in the transposon1545. The significantly increase of resistance may be closely related to ermB gene transfer that mediated by the transposon1545.After drug exposure, the expression of some virulence factors in Enterococcus faecalis were changed Significantly. The ratios of esp and cylA were elevated from0to70%, however the gelE ratio shows dramatically reducing from61.76%to20%. Meanwhile there are positive correlations between ermB and esp, cylA, whereas the relationship between the ermB and gelE was negative. As a consequence, we speculated that the resistance gene ermB and the virulence factors esp, cylA and gelE were located in the same accessoy gene region of Transposon1545. They were modulated by the same regulaors. Mediating by the Tn1545, the resistance gene and the virulence factors can transfer successfully in the intestinal flora.The toxicity of resistant Enterococcus faecalis including with virulence factors esp,cylA is more than the negative strains, after intraperitoneal injection, the former caused the death of the mice within8h, however, the latter is relative slow, mainly within24h, LD50of the latter is3.8times to the former. Under the adverse environment, E. faecalis harboring esp can have growth advantage in competitive landscape.it can participate in the formation of biofilm, be benefit in their attachment, settling and avoiding the elimination of host immune system.it can cause acute peritonitis and endocarditis, and induced bacteremia and sepsis, resulting in the death of the mice.At the same time, the risks of virulence increasing and resistance transfer in conditional pathogenic bacteria is also remarkable, except colonization resistance and resistance, caused by tulathromycin.