We have quantified the biological cost of VanA-type glycopeptide resistance due to the acquisition of the resistance operon by methicillin-resistant Staphylococcus aureus (MRSA) from Enterococcus sp. Exponential growths of recipient strain HIP11713, its transconjugant VRSA-1, VRSA-5, and VRSA-6 were compared in the absence or, except for HIP11713, in the presence of vancomycin. Induction of resistance was performed by adding vancomycin in both the preculture and the culture or the culture at only 1/50 the MIC. In the absence of vancomycin, the growth rates of the vancomycin-resistant S. aureus (VRSA) strains were similar to that of susceptible MRSA strain HIP11713. When resistance was induced, and under both conditions, there was a significant reduction of the growth rate of the VRSA strains relative to that of HIP11713 and to those of their noninduced counterparts, corresponding to a ca. 20% to 38% reduction in fitness. Competition experiments between isogenic VRSA-1 and HIP11713 mixed at a 1:1, 1:100, or 100:1 ratio revealed a competitive disadvantage of 0.4% to 3% per 10 generations of the transconjugant versus the recipient. This slight fitness burden can be attributed to the basal level of expression of the van genes in the absence of induction combined with a gene dosage effect due to the presence of the van operon on multicopy plasmids. These data indicate that VanA-type resistance, when induced, is highly costly for the MRSA host, whereas in the absence of induction, its biological cost is minimal. Thus, the potential for the dissemination of VRSA clinical isolates should not be underestimated.

Inducible vancomycin resistance in enterococci is due to a sophisticated mechanism that combines synthesis of cell wall peptidoglycan precursors with low affinity for glycopeptides and elimination of the normal target precursors. Although this dual mechanism, which involves seven genes organized in two operons, is predicted to have a high fitness cost, resistant enterococci have disseminated worldwide. We have evaluated the biological cost of VanB-type resistance due to acquisition of conjugative transposon Tn1549 in Enterococcus faecium and Enterococcus faecalis. Because fitness was dependent on the integration site of Tn1549, an isogenic set of E. faecalis was constructed to determine the cost of inducible or constitutive expression of resistance or of carriage of Tn1549. A luciferase gene was inserted in the integrase gene of the transposon to allow differential quantification of the strains in cocultures and in the digestive tract of gnotobiotic mice. Both in vitro and in vivo, carriage of inactivated or inducible Tn1549 had no cost for the host in the absence of induction by vancomycin. In contrast, induced or constitutively resistant strains not only had reduced fitness but were severely impaired in colonization ability and dissemination among mice. These data indicate that tight regulation of resistance expression drastically reduces the biological cost associated with vancomycin resistance in Enterococcus spp. and accounts for the widespread dissemination of these strains. Our findings are in agreement with the observation that regulation of expression is common in horizontally acquired resistance and represents an efficient evolutionary pathway for resistance determinants to become selectively neutral.O ne of the key parameters influencing emergence and stability of antibiotic resistance is the biological cost that resistance determinants impose on cell growth (1). Many studies have shown that resistant bacteria are less fit than their susceptible counterpart, although they can acquire compensatory mutations that restore fitness (2). However, most reports have dealt with resistance due to chromosomal mutations, and fewer have evaluated the fitness change due to acquisition of mobile genetic elements conferring clinically relevant resistance (3).Enterococci are commensals of the intestine of humans and animals but can also be opportunistic pathogens. This bacterial genus has acquired resistance to many antibiotics, and glycopeptides are often the ultimate treatment of infections due to these The vanA operon is part of transposon Tn1546, which is often carried by self-transferable plasmids, accounting for its spread (9). Dissemination of VanB-type resistance results from the spread of conjugative transposon Tn1549 (10) located on plasmids or in the chromosome. Despite the sophisticated dual mechanism of resistance, glycopeptide-resistant enterococci have disseminated worldwide.Two studies have investigated the burden of glycopeptide resistance imposed on enterococci, with conflicting results. A fitness di...

Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R 2 ‫؍‬ 0.98) or transcutaneously in the abdominal region of whole animals (R 2 ‫؍‬ 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2⍀P ami luxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.Among the wide variety of bacteria that colonize the gastrointestinal tracts of mammals, Escherichia coli is the most abundant facultative anaerobe of the human intestinal microflora. Aside from being part of the normal flora, E. coli is also a versatile organism capable of causing a variety of intestinal and extraintestinal diseases (18). The mechanisms that allow commensal E. coli to colonize the intestine and survive successfully in this niche remain poorly characterized. Conventional mice display natural resistance to colonization by commensal E. coli, but oral administration of streptomycin, which alters the intestinal microflora, allows colonization of the mouse large intestine by this species (25). The streptomycin-treated mouse model has been used extensively to study the factors of gramnegative bacteria implicated in the intestinal colonization process. However, this model is limited to the viable plate counts of bacteria in the feces and misses some critical information, such as the kinetics of colonization, the fate of the bacterial cells across the digestive tract, and the site of colonization. A better understanding of colonization would be facilitated by direct in vivo follow-up of this process.Biolumine...

Background-Daily administration of rectal formulations of mesalazine is eVective in preventing relapse of ulcerative proctitis. Maintenance of remission with lower doses would be an advantage. Aim-The eYcacy of mesalazine suppositories (Pentasa) 1 g three times a week v placebo to maintain remission in patients with cryptogenetic proctitis was studied. Methods-Ninety five patients with cryptogenetic proctitis were randomised within two weeks of remission to receive for one year or until relapse three suppositories per week of either Pentasa (n=48) or placebo (n=47). In the case of a relapse, the patients received one suppository/day. Results-It was found that 25 of 48 subjects v 18 of 47 remained in remission in the mesalazine and placebo groups respectively. The relapse rate was lower in the mesalazine group for the following time intervals: 0-90 days (19% v 38%, p=0.035), 0-180 days (29% v 54%, p=0.017), 0-270 days (38% v 60%, p=0.031), and 0-365 days (48% v 62%, p=0.18). Treatment of relapse with one suppository/day induced remission in 11 of 18 and 2 of 26 patients in the mesalazine and placebo groups respectively (p=0.001). Overall, 61% v 28% patients remained in the protocol and were in remission at one year (p=0.001). Tolerance was good. Conclusion-Mesalazine suppositories 1 g three times a week are eVective for preventing relapses of cryptogenetic proctitis. Increasing the dose to 1 g/day is eVective in a high proportion of subjects who relapsed.

We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 g/ml) and teicoplanin (MIC, 64 g/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 g/ml) and to low levels of teicoplanin (MIC, 4 g/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 g/ml) but susceptible to teicoplanin (MIC, 0.5 g/ml). (6,7,10,13,16,31), two E. faecalis strains (13), one Enterococcus gallinarum strain (8), and one Enterococcus raffinosus strain (37). Recently, the vanD operon was detected in a Ruminococcus sp., an anaerobic gram-positive bacterium from human bowel flora, suggesting a potential reservoir for vancomycin resistance genes in commensals of the digestive tract (17).VanD-type strains share various characteristics that distinguish them from VanA-and VanB-type enterococci. In particular, resistance to moderate levels of the two glycopeptides is constitutively expressed and is not transferable by conjugation to other enterococci (10,13,16,31). The organization of the vanD gene cluster, located exclusively in the chromosome, is similar to those of vanA and vanB (13,16