The selfish yeast plasmid exploits a SWI/SNF-type chromatin remodeling complex for hitchhiking on chromosomes and ensuring high-fidelity propagation.

Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STB bypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.

Disintegration promotes protospacer integration by the Cas1-Cas2 complex.

'Disintegration'-the reversal of transposon DNA integration at a target site-is regarded as an abortive off-pathway reaction. Here, we challenge this view with a biochemical investigation of the mechanism of protospacer insertion, which is mechanistically analogous to DNA transposition, by the Streptococcus pyogenes Cas1-Cas2 complex. In supercoiled target sites, the predominant outcome is the disintegration of one-ended insertions that fail to complete the second integration event. In linear target sites, one-ended insertions far outnumber complete protospacer insertions. The second insertion event is most often accompanied by the disintegration of the first, mediated either by the 3'-hydroxyl exposed during integration or by water. One-ended integration intermediates may mature into complete spacer insertions via DNA repair pathways that are also involved in transposon mobility. We propose that disintegration-promoted integration is functionally important in the adaptive phase of CRISPR-mediated bacterial immunity, and perhaps in other analogous transposition reactions.

The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning.

Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.

A bipartite thermodynamic-kinetic contribution by an activating mutation to RDF-independent excision by a phage serine integrase.

Streptomyces phage ϕC31 integrase (Int)-a large serine site-specific recombinase-is autonomous for phage integration (attP x attB recombination) but is dependent on the phage coded gp3, a recombination directionality factor (RDF), for prophage excision (attL x attR recombination). A previously described activating mutation, E449K, induces Int to perform attL x attR recombination in the absence of gp3, albeit with lower efficiency. E449K has no adverse effect on the competence of Int for attP x attB recombination. Int(E449K) resembles Int in gp3 mediated stimulation of attL x attR recombination and inhibition of attP x attB recombination. Using single-molecule analyses, we examined the mechanism by which E449K activates Int for gp3-independent attL x attR recombination. The contribution of E449K is both thermodynamic and kinetic. First, the mutation modulates the relative abundance of Int bound attL-attR site complexes, favoring pre-synaptic (PS) complexes over non-productively bound complexes. Roughly half of the synaptic complexes formed from Int(E449K) pre-synaptic complexes are recombination competent. By contrast, Int yields only inactive synapses. Second, E449K accelerates the dissociation of non-productively bound complexes and inactive synaptic complexes formed by Int. The extra opportunities afforded to Int(E499K) in reattempting synapse formation enhances the probability of success at fruitful synapsis.

A Flp-SUMO hybrid recombinase reveals multi-layered copy number control of a selfish DNA element through post-translational modification.

Mechanisms for highly efficient chromosome-associated equal segregation, and for maintenance of steady state copy number, are at the heart of the evolutionary success of the 2-micron plasmid as a stable multi-copy extra-chromosomal selfish DNA element present in the yeast nucleus. The Flp site-specific recombination system housed by the plasmid, which is central to plasmid copy number maintenance, is regulated at multiple levels. Transcription of the FLP gene is fine-tuned by the repressor function of the plasmid-coded partitioning proteins Rep1 and Rep2 and their antagonist Raf1, which is also plasmid-coded. In addition, the Flp protein is regulated by the host's post-translational modification machinery. Utilizing a Flp-SUMO fusion protein, which functionally mimics naturally sumoylated Flp, we demonstrate that the modification signals ubiquitination of Flp, followed by its proteasome-mediated degradation. Furthermore, reduced binding affinity and cooperativity of the modified Flp decrease its association with the plasmid FRT (Flp recombination target) sites, and/or increase its dissociation from them. The resulting attenuation of strand cleavage and recombination events safeguards against runaway increase in plasmid copy number, which is deleterious to the host-and indirectly-to the plasmid. These results have broader relevance to potential mechanisms by which selfish genomes minimize fitness conflicts with host genomes by holding in check the extra genetic load they pose.

Hitchhiking on chromosomes: A persistence strategy shared by diverse selfish DNA elements.

An underlying theme in the segregation of low-copy bacterial plasmids is the assembly of a 'segrosome' by DNA-protein and protein-protein interactions, followed by energy-driven directed movement. Analogous partitioning mechanisms drive the segregation of host chromosomes as well. Eukaryotic extra-chromosomal elements, exemplified by budding yeast plasmids and episomes of certain mammalian viruses, harbor partitioning systems that promote their physical association with chromosomes. In doing so, they indirectly take advantage of the spindle force that directs chromosome movement to opposite cell poles. Molecular-genetic, biochemical and cell biological studies have revealed several unsuspected aspects of 'chromosome hitchhiking' by the yeast 2-micron plasmid, including the ability of plasmid sisters to associate symmetrically with sister chromatids. As a result, the plasmid overcomes the 'mother bias' experienced by plasmids lacking a partitioning system, and elevates itself to near chromosome status in equal segregation. Chromosome association for stable propagation, without direct energy expenditure, may also be utilized by a small minority of bacterial plasmids-at least one case has been reported. Given the near perfect accuracy of chromosome segregation, it is not surprising that elements residing in evolutionarily distant host organisms have converged upon the common strategy of gaining passage to daughter cells as passengers on chromosomes.

Single-Molecule Tethered Particle Motion: Stepwise Analyses of Site-Specific DNA Recombination.

Tethered particle motion/microscopy (TPM) is a biophysical tool used to analyze changes in the effective length of a polymer, tethered at one end, under changing conditions. The tether length is measured indirectly by recording the Brownian motion amplitude of a bead attached to the other end. In the biological realm, DNA, whose interactions with proteins are often accompanied by apparent or real changes in length, has almost exclusively been the subject of TPM studies. TPM has been employed to study DNA bending, looping and wrapping, DNA compaction, high-order DNA⁻protein assembly, and protein translocation along DNA. Our TPM analyses have focused on tyrosine and serine site-specific recombinases. Their pre-chemical interactions with DNA cause reversible changes in DNA length, detectable by TPM. The chemical steps of recombination, depending on the substrate and the type of recombinase, may result in a permanent length change. Single molecule TPM time traces provide thermodynamic and kinetic information on each step of the recombination pathway. They reveal how mechanistically related recombinases may differ in their early commitment to recombination, reversibility of individual steps, and in the rate-limiting step of the reaction. They shed light on the pre-chemical roles of catalytic residues, and on the mechanisms by which accessory proteins regulate recombination directionality.

Sulfasalazine might reduce risk of cardiovascular diseases in patients with ankylosing spondylitis: A nationwide population-based retrospective cohort study.

AIM: To assess the effects of celecoxib and sulfasalazine on cardiovascular risk in patients with ankylosing spondylitis (AS). METHODS: We performed a 10-year population-based retrospective cohort study. A total of 1208 AS patients and 19 328 non-AS patients were sampled from the Taiwan National Health Insurance (NHI) database. We compared these two groups of patients to identify the differences in the exposure of non-steroidal anti-inflammatory drugs and sulfasalazine and their effects on cardiovascular risk. Univariate analyses were performed using Chi-squared tests for dichotomous variables and t-tests for continuous variables. Cox proportional hazard models were conducted to investigate the risk of developing cardiovascular diseases (CVD). RESULTS: AS patients had an adjusted hazard ratio (HR) of 1.72 (CI = 1.46-2.02, P < 0.01) for CVD compared with non-AS controls. The risk increased significantly with the progression of the disease. The use of celecoxib and sulfasalazine provided protective effects against CVD in both groups of patients. Both drugs at high cumulative defined daily doses (DDD) and celecoxib alone at high cumulative DDD showed significant protective effects against CVD in AS patients and the control group, respectively. Sulfasalazine at ≥ 0.5 DDD (1000 mg/day) reduced CVD risk in patients with AS (HR = 0.65, CI = 0.43-0.998, P < 0.05). CONCLUSIONS: In this population-based retrospective cohort study, sulfasalazine at its optimal dose reduced CVD risk in patients with AS. Celecoxib was neutral regarding CVD risk in AS patients.

Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion.

Serine and tyrosine site-specific recombinases (SRs and YRs, respectively) provide templates for understanding the chemical mechanisms and conformational dynamics of strand cleavage/exchange between DNA partners. Current evidence suggests a rather intriguing mechanism for serine recombination, in which one half of the cleaved synaptic complex undergoes a 180° rotation relative to the other. The 'small' and 'large' SRs contain a compact amino-terminal catalytic domain, but differ conspicuously in their carboxyl-terminal domains. So far, only one serine recombinase has been analyzed using single substrate molecules. We now utilized single-molecule tethered particle motion (TPM) to follow step-by-step recombination catalyzed by a large SR, phage ϕC31 integrase. The integrase promotes unidirectional DNA exchange between attB and attP sites to integrate the phage genome into the host chromosome. The recombination directionality factor (RDF; ϕC31 gp3) activates the excision reaction (attL × attR). From integrase-induced changes in TPM in the presence or absence of gp3, we delineated the individual steps of recombination and their kinetic features. The gp3 protein appears to regulate recombination directionality by selectively promoting or excluding active conformations of the synapse formed by specific att site partners. Our results support a 'gated rotation' of the synaptic complex between DNA cleavage and joining.

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome.

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts' fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates 'mother bias' (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation.

Simultaneous measurement of dynamic displacement and strain in a single fiber using coarse wavelength-division multiplexing and fiber Bragg-grating filter-based sensing system.

Displacement and strain, two of the most important physical quantities in experimental solid mechanics, are seldomly measured simultaneously in a single experimental configuration. In order to provide and improve corresponding sensing techniques, an experimental setup system for simultaneous measurement of dynamic displacement and strain on a flexible cantilever beam using two fiber Bragg gratings (FBGs) in a single fiber is proposed. To realize high-speed multiplexing and demodulation, a configuration incorporating a coarse wavelength-division multiplexing (CWDM) technique and an FBG transmission filter is implemented. The cantilever beam is subjected to steel-ball impact from which the dynamic multipoint displacement/strain sensing performances of the CWDM and FBG filter-based sensing system are demonstrated. Experimental results in temporal and frequency domain are compared with those obtained by the finite element method (FEM) predictions based on identification of the impact-loading history. A noncontact Fotonic displacement sensor and a polyvinylidene-fluoride film (PVDF) strain sensor are also used for comparison. With transient and resonant frequency simulations conducted by the FEM, loading effects of the sensing system are examined. The results obtained in this study indicate that the proposed CWDM and FBG filter-based sensing system is capable of performing simultaneous multipoint displacement/strain measurements in a single fiber with large bandwidth, high sensitivity, and low intensity loss.

Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase.

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR-HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.

Stable persistence of the yeast plasmid by hitchhiking on chromosomes during vegetative and germ-line divisions of host cells.

The chromosome-like stability of the Saccharomyces cerevisiae plasmid 2 micron circle likely stems from its ability to tether to chromosomes and segregate by a hitchhiking mechanism. The plasmid partitioning system, responsible for chromosome-coupled segregation, is comprised of 2 plasmid coded proteins Rep1 and Rep2 and a partitioning locus STB. The evidence for the hitchhiking model for mitotic plasmid segregation, although compelling, is almost entirely circumstantial. Direct tests for plasmid-chromosome association are hampered by the limited resolving power of current cell biological tools for analyzing yeast chromosomes. Recent investigations, exploiting the improved resolution of yeast meiotic chromosomes, have revealed the plasmid's propensity to be present at or near chromosome tips. This localization is consistent with the rapid plasmid movements during meiosis I prophase, closely resembling telomere dynamics driven by a meiosis-specific nuclear envelope motor. Current evidence is consistent with the plasmid utilizing the motor as a platform for gaining access to telomeres. Episomes of viruses of the papilloma family and the gammaherpes subfamily persist in latently infected cells by tethering to chromosomes. Selfish genetic elements from fungi to mammals appear to have, by convergent evolution, arrived at the common strategy of chromosome association as a means for stable propagation.

An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective.

Tyrosine site-specific recombinases (YRs) are widely distributed among prokaryotes and their viruses, and were thought to be confined to the budding yeast lineage among eukaryotes. However, YR-harboring retrotransposons (the DIRS and PAT families) and DNA transposons (Cryptons) have been identified in a variety of eukaryotes. The YRs utilize a common chemical mechanism, analogous to that of type IB topoisomerases, to bring about a plethora of genetic rearrangements with important physiological consequences in their respective biological contexts. A subset of the tyrosine recombinases has provided model systems for analyzing the chemical mechanisms and conformational features of the recombination reaction using chemical, biochemical, topological, structural, and single molecule-biophysical approaches. YRs with simple reaction requirements have been utilized to bring about programmed DNA rearrangements for addressing fundamental questions in developmental biology. They have also been employed to trace the topological features of DNA within high-order DNA interactions established by protein machines. The directed evolution of altered specificity YRs, combined with their spatially and temporally regulated expression, heralds their emergence as vital tools in genome engineering projects with wide-ranging biotechnological and medical applications.

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of 'non-productive', 'pre-synaptic' and 'synaptic' complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations.

The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells.

The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications.

Investigation of polyvinylidene fluoride (PVDF) films in identifying high-frequency vibration modes of flexible plates.

Compared with piezoelectric ceramics such as lead zirconate titanate (PZT) ceramics, the low density and high compliance of the PVDF films make them a more suitable choice in modal testing, especially for detecting high-frequency modes in flexible or inflatable structures. In this work, dynamic sensing performances of PVDF films for flexible structures in modal testing are examined, with considerations including the repeatability of the impact source, the accuracy of the sensing responses, and the influences of the nodal lines on the frequency spectra of the transient responses. Two flexible plates with different boundary conditions and thickness are considered. Experimental results, compared with FEM computations or theoretical predictions, demonstrate the excellent dynamic sensing performance of the PVDF film in modal testing applications, especially for identification of high-frequency modes on flexible structures.

Organization of DNA partners and strand exchange mechanisms during Flp site-specific recombination analyzed by difference topology, single molecule FRET and single molecule TPM.

Flp site-specific recombination between two target sites (FRTs) harboring non-homology within the strand exchange region does not yield stable recombinant products. In negatively supercoiled plasmids containing head-to-tail sites, the reaction produces a series of knots with odd-numbered crossings. When the sites are in head-to-head orientation, the knot products contain even-numbered crossings. Both types of knots retain parental DNA configuration. By carrying out Flp recombination after first assembling the topologically well defined Tn3 resolvase synapse, it is possible to determine whether these knots arise by a processive or a dissociative mechanism. The nearly exclusive products from head-to-head and head-to-tail oriented "non-homologous" FRT partners are a 4-noded knot and a 5-noded knot, respectively. The corresponding products from a pair of native (homologous) FRT sites are a 3-noded knot and a 4-noded catenane, respectively. These results are consistent with non-homology-induced two rounds of dissociative recombination by Flp, the first to generate reciprocal recombinants containing non-complementary base pairs and the second to produce parental molecules with restored base pairing. Single molecule fluorescence resonance energy transfer (smFRET) analysis of geometrically restricted FRTs, together with single molecule tethered particle motion (smTPM) assays of unconstrained FRTs, suggests that the sites are preferentially synapsed in an anti-parallel fashion. This selectivity in synapse geometry occurs prior to the chemical steps of recombination, signifying early commitment to a productive reaction path. The cumulative topological, smFRET and smTPM results have implications for the relative orientation of DNA partners and the directionality of strand exchange during recombination mediated by tyrosine site-specific recombinases.

Prospective, randomized, study of ampicillin-sulbactam versus moxifloxacin monotherapy for the treatment of community-acquired complicated intra-abdominal infections.

BACKGROUND: The ideal antimicrobial treatment for intra-abdominal infections (IAIs) in the setting of fast-paced emergency departments (EDs) should be effective, convenient, and of limited resource utilization. Antibiotic monotherapy is a feasible option for this. We conducted a study in which we compared two regimens for antibiotic monotherapy recommended by published guidelines in ED patients with community-acquired, complicated IAIs (cIAIs). METHODS: The study was a prospective, randomized, study of ampicillin-sulbactam versus moxifloxacin for cIAIs. After the diagnosis of cIAI was established, patients were assigned randomly to receive either moxifloxacin 400 mg intravenously (IV) qd followed by moxifloxacin 400 mg orally (PO) qd, or ampicillin-sulbactam 1.5 g IV qid followed by ampicillin-sulbactam 750 mg PO q12h. Source control procedures were used for all patients and all had complete follow-up. The primary efficacy variable for the study was the clinical response at the test-of-cure visit. RESULTS: A total of 116 patients were enrolled for prospective evaluation and randomized assignment to treatment with ampicillin-sulbactam (n=55) or moxifloxacin (n=61). At the test-of-cure evaluation, the overall clinical failure rate was 13.8%. The clinical failure rates in the ampicillin-sulbactam and moxifloxacin groups were 16.4% (9/55) and 11.5% (7/61), respectively (p=0.446). With regard to infection site, the clinical failure rate in cIAIs consisting of lower gastrointestinal (GI) tract infection was significantly lower in the moxifloxacin than in the ampicillin-sulbactam group (4.3% vs. 19.6%; p=0.024). According to multivariable analysis, independent risk factors for treatment failure were the time to ED presentation >24 h (odds ratio [OR] 6.8; 95% CI 1.3-36.2; p=0.024) and ampicillin-sulbactam therapy (OR 9.5; 95% CI 1.1-76.6; p=0.033). CONCLUSIONS: A significant difference existed in the clinical responses of the two groups. As compared with ampicillin-sulbactam, moxifloxacin was more effective for the treatment of community-acquired cIAIs of the lower GI tract. A higher risk of treatment failure for antibiotic therapy was found for patients presenting to the ED with symptoms of cIAIs lasting >24 h. Alternative antimicrobial agents should be considered for treating these patients.