The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable, but can nevertheless be controlled and must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of thirty scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, May 16-18, 2011. From these discussions emerged a priority list of steps that need to be taken to resolve this global crisis.

Daptomycin is the first available agent from a new class of antibiotics, the cyclic lipopeptides, that has activity against a broad range of gram-positive pathogens, including organisms that are resistant to methicillin, vancomycin, and other currently available agents. Daptomycin (4 mg/kg intravenously [iv] every 24 h for 7-14 days) was compared with conventional antibiotics (penicillinase-resistant penicillins [4-12 g iv per day] or vancomycin [1 g iv every 12 h]) in 2 randomized, international trials involving 1092 patients with complicated skin and skin-structure infections. Among 902 clinically evaluable patients, clinical success rates were 83.4% and 84.2% for the daptomycin- and comparator-treated groups, respectively (95% confidence interval, -4.0 to 5.6). Among patients successfully treated with iv daptomycin, 63% required only 4-7 days of therapy, compared with 33% of comparator-treated patients (P<.0001). The frequency and distribution of adverse events were similar among both treatment groups. Overall, the safety and efficacy of daptomycin were comparable with conventional therapy.

The expression of type 1 ribbriae (pili) of Escherichia coli is turned on and off at the transcriptional level at a high frequency (10-3 per cell per generation) in a process termed phase variation. Using Southern blot and DNA sequence analysis, we have detected a genomic rearrangement in the switch region immediately upstream of the fimbrial structural gene. This rearrangement involves an invertible 314-basepair segment of DNA whose alternating orientation apparently results in the on-and-off activation of a promoter that determines the state of fimbrial expression.Type 1 fimbriae (pili) are major surface appendages that mediate binding of Escherichia coli to eukaryotic cells by a ligand-receptor mechanism that is sensitive to the presence of mannose. The fimbrial protein adhesin is thought to be a virulence factor during the initial colonization stage. During the invasive stage it would seem to be to the pathogen's advantage not to express adhesins that could mediate binding to phagocytic cells. A genetic regulatory system that would result in an on-and-off expression of an adhesin would itself be a virulence factor (1).The phase variation between fimbriate (Fim+) and nonfimbriate (Fim-) E. coli cells occurs at the transcriptional level (2). In addition, it has been shown that a cis-dominant DNA switch is turned on and off by means of a trans-active factor (3) different from that reported by Orndorff and Falkow (4). Here we report that the molecular basis of this switch is the inversion of a relatively small segment of DNA that results in the alternating activation of the fimbrial promoter in correlation with phase variation. This system resembles in general that which controls flagellar phase variation in Salmonella (5) but differs both in its recombinase specificity and in its small size, so that the element does not encode its own recombinase. MATERIALS AND METHODS Bacterial Strains and Media. E. coli K-12 strain CSH50[ara A(lac-pro) rpsL thi] was the parental strain used. The genetic construction of strain VL412 and bacteriophage X412 has been presented in detail (3). 5-Bromo-4-chloro-3-indolyl ,B-D-galactopyranoside (X-Gal) RESULTSThe Lac-E. coli K-12 strain CSH50, used in the genetic constructions outlined below, undergoes fimbrial phase variation. Eisenstein (2) described the construction of afim-lac operon fusion in this strain and used its oscillating Lac';±Lac-phenotype to show that phase variation is under transcriptional control (2). The fim', fim-lac merodiploid strain VL412 (Fig. 1) was constructed through integration of a specialized X phage, carrying the fim-lac operon fusion, by homologous recombination with the fimD gene (3). This merodiploid strain exhibits independent, reversible, and noncoordinated phase variation of both the Lac and Fim phenotypes, which indicates the presence of two cis-acting switches, each adjacent to its respective operon (3). Ultraviolet induction of VL412 yields the phage X412, which alternates between a very dark blue (Dk) and light blue (Lt) plaque phe...

To facilitate efficient allelic exchange of genetic information into a wild-type strain background, we improved upon and merged approaches using a temperature-sensitive plasmid and a counter-selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild-type chromosomal sequences, at either the fimB-A or lacZ-A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn5, but, unlike another similar cassette, it lacks IS1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature-sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non-selective conditions at 42 degrees C, and then in the presence of sucrose at 30 degrees C, allowing positive selection for both plasmid excision and curing. Despite marked locus-specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non-essential parts of the E. coli chromosome.

Expression of type 1 fimbriae in Escherichia coli K-12 is phase variable and associated with the inversion of a short DNA element (switch). Thefim switch requires eitherfimB (on-to-off or off-to-on switching) orfimE (on-to-off switching only) and is affected by the global regulators leucine-responsive regulatory protein (Lrp), integration host factor (IHF), and H-NS. Here it is shown that switching frequencies are regulated by both temperature and media and that these effects appear to be independent. fimE-promoted on-to-off switching occurs far more rapidly than previously estimated (0.3 per cell per generation in defined rich medium at 37°C) and faster at lower than at higher temperatures. In direct contrast, fimB-promoted switching increases with temperature, with optima between 37 and 41°C. Switching promoted by both fimB andfimE is stimulated by aliphatic amino acids (alanine, isoleucine, leucine, and valine), and this stimulation requires lrp. Furthermore, lrp appears to differentially regulate fimB-andfimE-promoted switching in different media.Type 1 fimbriae are filamentous proteinaceous appendages produced by many species of enteric bacteria. Type 1 fimbriae promote attachment to a variety of eukaryotic cells by a process inhibited by mannose (24). Recent evidence suggests that type 1 fimbriae play an important role in communicability (3). The expression of type 1 fimbriae may play a role in urinary tract infections (24). However, type 1 fimbriae are excellent immunogens (10,28,39,40). Thus, the capacity to switch rapidly their expression off or on, particularly in response to specific signals, should be advantageous to the organism and consequently important in pathogenesis.Type 1 fimbrial phase variation is associated with the inversion of a 314-bp DNA element (1). This switch (invertible element) contains a promoter forfimA, the main fimbrial structural subunit gene (16). Thus, fimA is expressed in one orientation (on) but not the other (off). Switching is RecA independent (site specific) and requires eitherfimB (on-to-off or off-to-on switching) or fimE (on-to-off switching only) (6,17,27,32,33,38). In addition to fimB and fimE, genes situated adjacent to the switch, switching is also influenced by at least three global regulators, leucine-responsive regulatory protein (Lrp), H-NS, and integration host factor (IHF) (4,11,15,21,26,45). Recently, we and others have shown that many K-12 strains studied are fimE mutants and that slow switching results from these mutations (6). Preliminary analysis of fimE+ K-12 strains demonstrated that on-to-off switching is much faster (at least 0.01 per cell per generation) than fimB-promoted switching (10-3 to 10-) (4, 6). Mutations in lrp (Lrp) and himA and himD (IHF) markedly reduce both fimB-and fimE-promoted switching (4,11,15 background (21,26,45). Interestingly, others have noted heterogeneity in control of phase variation among clinical isolates (2,18,22,23,41,42) presumably reflecting differences in the genetic compositions of these strains.The fim switch in...

An operon fusion of the lac genes to those required for synthesis of type 1 fimbriae (pili) has been achieved in a K12 strain of Escherichia coli lysogenized by the bacteriophage mu d (Ap4, lac). Synthesis of beta-galactosidase, therefore, reflected pil gene transcription and was used as a probe of fimbrial regulation. Expression of the operon fusion was found to oscillate, demonstrating that phase variation between fimbriate and nonfimbriate states is under transcriptional control. The transition rates from fimbriate to nonfimbriate were 1.05 X 10(-3) per bacterium per generation and from nonfimbriate to fimbriate, 3.12 X 10(-3) per bacterium per generation.

Evidence obtained with an improved in vivo assay of fimbrial phase variation in Escherichia coil supported a revised understanding of the roles offimB andfimE in the site-specific DNA rearrangement with which they are associated. A previously proposed model argued thatfimB andfimE play antagonistic, unidirectional roles in regulating the orientation of the invertible DNA element located immediately upstream offimA, the gene encoding the major subunit of type 1 fimbriae. This conclusion, though, is based on an in vivo DNA inversion assay using recombinant plasmid substrates under conditions that, among other things, were incapable of detecting recombination of thefim invertible element from the on to the off orientation. Using a modified system that overcame this and several additional technical problems, we confirmed thatflmB acts independently of fimE on the invertible element and that the additional presence offimE results in the preferential rearrangement of the element to the off orientation. It is now demonstrated that fimE can act in the absence offimB in this recombination to promote inversion primarily from on to off. In contrast to the previous studies, the effect offfimB on a substrate carrying the invertible element in the on orientation could be examined. It was found that fimB mediates DNA inversion from on to off, as well as from off to on, and that, contrary to prior interpretations, the flimB-associated inversion occurs with only minimal orientational preference to the on phase.Type 1 fimbriae, produced by many members of the family Enterobacteriaceae, are multisubunit proteinaceous appendages that mediate mannose-sensitive bacterial adherence to eukaryotic cells. In Escherichia coli, these organelles have been implicated in promoting colonization and subsequent infection. The genes encoding type 1 fimbriae in E. coli are positioned at 98 minutes on the chromosomal map (8). The oscillating on-and-off expression of the organelles, a process known as phase variation, is correlated with the inversion of a 314-bp DNA sequence immediately upstream offimA (1, 5). Type 1 fimbriae are produced when the invertible element is in one orientation (on) and are not produced when the invertible element is in the alternate orientation (off), presumably because of the presence of a promoter within the invertible element.A number of genes have been identified whose products affect the invertible element in trans, several mapping at sites distant from fim. One such gene, pilG (osmZ) (20, 32), recently shown to encode the histonelike protein HI (21, 25), alters the frequency of inversion by an unknown mechanism. Two other genes, himA and himDlhip, which together encode integration host factor (IHF), are required for efficient recombination of the fim invertible element (11,15). IHF is a heterodimeric DNA-binding and -bending protein which was initially identified for its role in bacteriophage lambda integration and excision (reviewed in reference 17). Two additional genes required for the inversion event, fimB and f...

To investigate the pathogenesis of Legionnaires disease at a molecular level, we mutated by directed allelic exchange a gene encoding a Legionella pneumophila-specific 24,000-dalton (Da) surface protein. Southern hybridization and immunoblot analyses demonstrated that the predicted DNA rearrangement occurred in L. pneumophila with a specific loss of 24-kDa antigen expression. Compared with its isogenic parent, the mutant was significantly impaired in its ability to infect transformed U937 cells, a human macrophagelike cell line; i.e., the bacterial inoculum of the mutant strain that was required to initiate infection of the macrophage monolayer was ca. 80-fold greater than that of the isogenic parent strain. The mutant strain regained full infectivity on reintroduction of a cloned 24-kDa protein gene, indicating that the reduced infectivity was due specifically to the mutation in that gene. Compared with the parent strain, the mutant strain was recovered at titers that were ca. 10-fold lower shortly after infection, but it exhibited a similar intracellular growth rate over the next 40 h, indicating that the mutant was defective in its ability to initiate macrophage infection rather than in its ability to replicate intraceilularly. When opsonized, the mutant strain was still significantly less infectious than the parent strain, despite equivalent macrophage association, suggesting that the mutant was not merely missing a ligand for macrophage attachment. The mutant also exhibited reduced infectivity in explanted human alveolar macrophages, demonstrating the relevance of the U937 cell model for analyzing this mutant phenotype. These results represent the first identification of a cloned L. pneumophila gene that is necessary for optimal intracellular infection; we designate this gene mip, for macrophage infectivity potentiator.