Structural basis for mechanical force regulation of the adhesin FimH via finger trap-like beta sheet twisting.

The Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multiprotein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the beta sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of the lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around the ligand like a finger-trap toy. Thus, beta sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.

Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.

Pandemic fluoroquinolone resistant Escherichia coli clone ST1193 emerged via simultaneous homologous recombinations in 11 gene loci.

Global growth in antibiotic resistance is a major social problem. A high level of resistance to fluoroquinolones requires the concurrent presence of at least 3 mutations in the target proteins-2 in DNA gyrase (GyrA) and 1 in topoisomerase IV (ParC), which occur in a stepwise manner. In the Escherichia coli chromosome, the gyrA and parC loci are positioned about 1 Mb away from each other. Here we show that the 3 fluoroquinolone resistance mutations are tightly associated genetically in naturally occurring strains. In the latest pandemic uropathogenic and multidrug-resistant E. coli clonal group ST1193, the mutant variants of gyrA and parC were acquired not by a typical gradual, stepwise evolution but all at once. This happened as part of 11 simultaneous homologous recombination events involving 2 phylogenetically distant strains of E. coli, from an uropathogenic clonal complex ST14 and fluoroquinolone-resistant ST10. The gene exchanges swapped regions between 0.5 and 139 Kb in length (183 Kb total) spread along 976 Kb of chromosomal DNA around and between gyrA and parC loci. As a result, all 3 fluoroquinolone resistance mutations in GyrA and ParC have simultaneously appeared in ST1193. Based on molecular clock estimates, this potentially happened as recently as <12 y ago. Thus, naturally occurring homologous recombination events between 2 strains can involve numerous chromosomal gene locations simultaneously, resulting in the transfer of distant but tightly associated genetic mutations and emergence of a both highly pathogenic and antibiotic-resistant strain with a rapid global spread capability.

Strain and host-cell dependent role of type-1 fimbriae in the adherence phenotype of super-shed Escherichia coli O157:H7.

Super-shed (SS) Escherichia coli O157 (E. coli O157) demonstrate a strong, aggregative, locus of enterocyte effacement (LEE)-independent adherence phenotype on bovine recto-anal junction squamous epithelial (RSE) cells, and harbor polymorphisms in non-LEE-adherence-related loci, including in the type 1 fimbriae operon. To elucidate the role of type 1 fimbriae in strain- and host-specific adherence, we evaluated the entire Fim operon (FimB-H) and its adhesion (FimH) deletion mutants in four E. coli O157 strains, SS17, SS52, SS77 and EDL933, and evaluated the adherence phenotype in bovine RSE and human HEp-2 adherence assays. Consistent with the prevailing dogma that fimH expression is genetically switched off in E. coli O157, the ΔfimHSS52, ΔfimB-HSS52, ΔfimB-HSS17, and ΔfimHSS77 mutants remained unchanged in adherence phenotype to RSE cells. In contrast, the ΔfimHSS17 and ΔfimB-HSS77 mutants changed from a wild-type strong and aggregative, to a moderate and diffuse adherence phenotype, while both ΔfimHEDL933 and ΔfimB-HEDL933 mutants demonstrated enhanced binding to RSE cells (p < 0.05). Additionally, both ΔfimHSS17 and ΔfimHEDL933 were non-adherent to HEp-2 cells (p < 0.05). Complementation of the mutant strains with their respective wild-type genes restored parental phenotypes. Microscopy revealed that the SS17 and EDL933 strains indeed carry type 1 fimbriae-like structures shorter than those seen in uropathogenic E. coli. Taken together, these results provide compelling evidence for a strain and host cell type-dependent role of fimH and the fim operon in E. coli O157 adherence that needs to be further evaluated.

RMSD analysis of structures of the bacterial protein FimH identifies five conformations of its lectin domain.

FimH is a bacterial adhesin protein located at the tip of Escherichia coli fimbria that functions to adhere bacteria to host cells. Thus, FimH is a critical factor in bacterial infections such as urinary tract infections and is of interest in drug development. It is also involved in vaccine development and as a model for understanding shear-enhanced catch bond cell adhesion. To date, over 60 structures have been deposited in the Protein Data Bank showing interactions between FimH and mannose ligands, potential inhibitors, and other fimbrial proteins. In addition to providing insights about ligand recognition and fimbrial assembly, these structures provide insights into conformational changes in the two domains of FimH that are critical for its function. To gain further insights into these structural changes, we have superposed FimH's mannose binding lectin domain in all these structures and categorized the structures into five groups of lectin domain conformers using RMSD as a metric. Many structures also include the pilin domain, which anchors FimH to the fimbriae and regulates the conformation and function of the lectin domain. For these structures, we have also compared the relative orientations of the two domains. These structural analyses enhance our understanding of the conformational changes associated with FimH ligand binding and domain-domain interactions, including its catch bond behavior through allosteric action of force in bacterial adhesion.

Pandemic Uropathogenic Fluoroquinolone-resistant Escherichia coli Have Enhanced Ability to Persist in the Gut and Cause Bacteriuria in Healthy Women.

We report that fluoroquinolone-resistant Escherichia coli are found in feces of 8.8% of healthy women, with most bacteria belonging to pandemic multidrug-resistant ST131-H30R or ST1193 clonal groups. Moreover, these highly uropathogenic clonal groups demonstrate an especially prolonged gut persistence and high rate of bacteriuria without documented urinary tract infection.

The Uropathogenic Escherichia coli Subclone Sequence Type 131-H30 Is Responsible for Most Antibiotic Prescription Errors at an Urgent Care Clinic.

BACKGROUND: The pandemic spread of antibiotic resistance increases the likelihood of ineffective empirical therapy. The recently emerged fluoroquinolone-resistant Escherichia coli sequence type (ST) 131-H30R subclone (H30) is a leading cause of multidrug-resistant urinary tract infection (UTI) and bloodstream infection worldwide. METHODS: We studied the relative impact of H30 on the likelihood that bacteria isolated from urine of urgent care patients would be resistant to the empirically prescribed antibiotic regimen for UTI. RESULTS: Of 750 urinalysis-positive urine samples from urgent care patients with suspected UTI, 306 (41%) yielded E. coli, from 35 different clonal groups (clonotypes). H30 predominated (14% prevalence overall), especially among older patients (age ≥70 years: 26%) and those with diabetes (43%) or urinary catheterization (60%). Resistance to the empirically selected antibiotic regimen occurred in 16% (40/246) of patients overall, 28% (20/71) of older patients, 30% (8/27) of patients with diabetes, 60% (3/5) of catheterized patients, and 71% (22/30) of those with H30. H30's contribution to such mismatched antibiotic selection was 55% overall, 70% among older patients, and 100% among patients with diabetes or a urinary catheter. Among patients with ≥2 of these factors (older age, diabetes, or urinary catheter), 24% of all urinalysis-positive urine samples yielded H30, with a 92% likelihood of resistance to the selected empirical therapy. CONCLUSIONS: The multidrug-resistant H30 subclone of E. coli ST131 is responsible for the great majority of mismatched empirical antibiotic prescriptions for suspected UTI at an urgent care clinic among patients ≥70 years old or with diabetes or urinary catheterization.

Rapid and Extensive Expansion in the United States of a New Multidrug-resistant Escherichia coli Clonal Group, Sequence Type 1193.

We describe the rapid and ongoing emergence across multiple US cities of a new multidrug-resistant Escherichia coli clone-sequence type (ST) 1193-resistant to fluoroquinolones (100%), trimethoprim-sulfamethoxazole (55%), and tetracycline (53%). ST1193 is associated with younger adults (age <40 years) and currently comprises a quarter of fluoroquinolone-resistant clinical E. coli urine isolates.

Escherichia coli Clonobiome: Assessing the Strain Diversity in Feces and Urine by Deep Amplicon Sequencing.

While microbiome studies have focused on diversity at the species level or higher, bacterial species in microbiomes are represented by different, often multiple, strains. These strains could be clonally and phenotypically very different, making assessment of strain content vital to a full understanding of microbiome function. This is especially important with respect to antibiotic-resistant strains, the clonal spread of which may be dependent on competition between them and susceptible strains from the same species. The pandemic, multidrug-resistant, and highly pathogenic Escherichia coli subclone ST131-H30 (H30) is of special interest, as it has already been found persisting in the gut and bladder in healthy people. In order to rapidly assess E. coli clonal diversity, we developed a novel method based on deep sequencing of two loci used for sequence typing, along with an algorithm for analysis of the resulting data. Using this method, we assessed fecal and urinary samples from healthy women carrying H30 and were able to uncover considerable diversity, including strains with frequencies at <1% of the E. coli population. We also found that, even in the absence of antibiotic use, H30 could completely dominate the gut and, especially, urine of healthy carriers. Our study offers a novel tool for assessing a species' clonal diversity (clonobiome) within the microbiome, which could be useful in studying the population structure and dynamics of multidrug-resistant and/or highly pathogenic strains in their natural environments.IMPORTANCE Bacterial species in the microbiome are often represented by multiple genetically and phenotypically different strains, making insight into subspecies diversity critical to a full understanding of the microbiome, especially with respect to opportunistic pathogens. However, methods allowing efficient high-throughput clonal typing are not currently available. This study combines a conventional E. coli typing method with deep amplicon sequencing to allow analysis of many samples concurrently. While our method was developed for E. coli, it may be adapted for other species, allowing microbiome researchers to assess clonal strain diversity in natural samples. Since assessment of subspecies diversity is particularly important for understanding the spread of antibiotic resistance, we applied our method to the study of a pandemic multidrug-resistant E. coli clone. The results we present suggest that this clone could be highly competitive in healthy carriers and that the mechanisms of colonization by such clones need to be studied.

Epidemiology and Antimicrobial Resistance Characteristics of the Sequence Type 131-H30 Subclone Among Extraintestinal Escherichia coli Collected From US Children.

Background: Escherichia coli sequence type (ST) 131-H30 is a globally important pathogen implicated in rising rates of multidrug resistance among E. coli causing extraintestinal infections. Previous studies have focused on adults, leaving the epidemiology of H30 among children undefined. Methods: We used clinical data and isolates from a case-control study of extended-spectrum cephalosporin-resistant E. coli conducted at 4 US children's hospitals to estimate the burden and identify host correlates of infection with H30. H30 isolates were identified using 2-locus genotyping; host correlates were examined using log-binomial regression models stratified by extended-spectrum cephalosporin resistance status. Results: A total of 339 extended-spectrum cephalosporin-resistant and 1008 extended-spectrum cephalosporin-susceptible E. coli isolates were available for analyses. The estimated period prevalence of H30 was 5.3% among all extraintestinal E. coli isolates (95% confidence interval [CI], 4.6%-7.1%); H30 made up 43.3% (81/187) of extended-spectrum ß-lactamase (ESBL)-producing isolates in this study. Host correlates of infection with H30 differed by extended-spectrum cephalosporin resistance status: Among resistant isolates, age ≤5 years was positively associated with H30 infection (relative risk [RR], 1.83 [95% CI, 1.19-2.83]); among susceptible isolates, age ≤5 years was negatively associated with H30 (RR, 0.48 [95% CI, .27-.87]), while presence of an underlying medical condition was positively associated (RR, 4.49 [95% CI, 2.43-8.31]). Conclusions: ST131-H30 is less common among extraintestinal E. coli collected from children compared to reported estimates among adults, possibly reflecting infrequent fluoroquinolone use in pediatrics; however, it is similarly dominant among ESBL-producing isolates. The H30 subclone appears to disproportionately affect young children relative to other extended-spectrum cephalosporin-resistant E. coli.

Recombination-independent rapid convergent evolution of the gastric pathogen Helicobacter pylori.

BACKGROUND: Helicobacter pylori is a human stomach pathogen, naturally-competent for DNA uptake, and prone to homologous recombination. Extensive homoplasy (i.e., phylogenetically-unlinked identical variations) observed in H. pylori genes is considered a hallmark of such recombination. However, H. pylori also exhibits a high mutation rate. The relative adaptive role of homologous recombination and mutation in species diversity is a highly-debated issue in biology. Recombination results in homoplasy. While convergent mutation can also account for homoplasy, its contribution is thought to be minor. We demonstrate here that, contrary to dogma, convergent mutation is a key contributor to Helicobacter pylori homoplasy, potentially driven by adaptive evolution of proteins. RESULTS: Our present genome-wide analysis shows that homoplastic nonsynonymous (amino acid replacement) changes are not typically accompanied by homoplastic synonymous (silent) variations. Moreover, the majority of the codon positions with homoplastic nonsynonymous changes also contain different (i.e. non-homoplastic) nonsynonymous changes arising from mutation only. This indicates that, to a considerable extent, nonsynonymous homoplasy is due to convergent mutations. High mutation rate or limited availability of evolvable sites cannot explain this excessive convergence, as suggested by our simulation studies. Rather, the genes with convergent mutations are overrepresented in distinct functional categories, suggesting possible selective responses to conditions such as distinct micro-niches in single hosts, and to differences in host genotype, physiology, habitat and diet. CONCLUSIONS: We propose that mutational convergence is a key player in H. pylori's adaptation and extraordinary persistence in human hosts. High frequency of mutational convergence could be due to saturation of evolvable sites capable of responding to selection pressures, while the number of mutable residues is far from saturation. We anticipate a similar scenario of mutational vs. recombinational genome dynamics or plasticity for other naturally competent microbes where strong positive selection could favor frequent convergent mutations in adaptive protein evolution.

Inactive conformation enhances binding function in physiological conditions.

Many receptors display conformational flexibility, in which the binding pocket has an open inactive conformation in the absence of ligand and a tight active conformation when bound to ligand. Here we study the bacterial adhesin FimH to address the role of the inactive conformation of the pocket for initiating binding by comparing two variants: a wild-type FimH variant that is in the inactive state when not bound to its target mannose, and an engineered activated variant that is always in the active state. Not surprisingly, activated FimH has a longer lifetime and higher affinity, and bacteria expressing activated FimH bound better in static conditions. However, bacteria expressing wild-type FimH bound better in flow. Wild-type and activated FimH demonstrated similar mechanical strength, likely because mechanical force induces the active state in wild-type FimH. However, wild-type FimH displayed a faster bond association rate than activated FimH. Moreover, the ability of different FimH variants to mediate adhesion in flow reflected the fraction of FimH in the inactive state. These results demonstrate a new model for ligand-associated conformational changes that we call the kinetic-selection model, in which ligand-binding selects the faster-binding inactive state and then induces the active state. This model predicts that in physiological conditions for cell adhesion, mechanical force will drive a nonequilibrium cycle that uses the fast binding rate of the inactive state and slow unbinding rate of the active state, for a higher effective affinity than is possible at equilibrium.

Inactivation of Transcriptional Regulators during Within-Household Evolution of Escherichia coli.

We analyzed the within-household evolution of two household-associated Escherichia coli strains from pandemic clonal group ST131-H30, using isolates recovered from five individuals within two families, each of which had a distinct strain. Family 1's strain was represented by a urine isolate from the index patient (older sister) with recurrent cystitis and a blood isolate from her younger sister with fatal urosepsis. Family 2's strain was represented by a urine isolate from the index patient (father) with pyelonephritis and renal abscesses, blood and kidney drainage isolates from the daughter with emphysematous pyelonephritis, and urine and fecal isolates from the mother with cystitis. Collectively, the several variants of each family's strain had accumulated a total of 8 (family 1) and 39 (family 2) point mutations; no two isolates were identical. Of the 47 total mutations, 36 resulted in amino acid changes or truncation of coded proteins. Fourteen such mutations (39%) targeted genes encoding transcriptional regulators, and 9 (25%) involved DNA-binding transcription factors (TFs), which significantly exceeded the relative contribution of TF genes to the isolates' genomes (∼6%). At least one-half of the transcriptional regulator mutations were inactivating, based on phenotypic and/or transcriptional analysis. In particular, inactivating mutations in the global regulator LrhA (repressor of type 1 fimbriae and flagella) occurred in the blood isolates from both households and increased the virulence of E. coli strains in a murine sepsis model. The results indicate that E. coli undergoes adaptive evolution between and/or within hosts, generating subpopulations with distinctive phenotypes and virulence potential.IMPORTANCE The clonal evolution of bacterial strains associated with interhost transmission is poorly understood. We characterized the genome sequences of clonal descendants of two Escherichia coli strains, recovered at different time points from multiple individuals within two households who had different types of urinary tract infection. We found evidence that the E. coli strains underwent extensive mutational diversification between and within these individuals, driven disproportionately by inactivation of transcriptional regulators. In urosepsis isolates, the mutations observed in the global regulator LrhA increased bacterial virulence in a murine sepsis model. Our findings help in understanding the adaptive dynamics and strategies of E. coli during short-term natural evolution.

Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic E. coli.

Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.

Corrected Genome Annotations Reveal Gene Loss and Antibiotic Resistance as Drivers in the Fitness Evolution of Salmonella enterica Serovar Typhimurium.

Horizontal acquisition of novel chromosomal genes is considered to be a key process in the evolution of bacterial pathogens. However, the identification of gene presence or absence could be hindered by the inconsistencies in bacterial genome annotations. Here, we performed a cross-annotation of omnipresent core and mosaic accessory genes in the chromosome of Salmonella enterica serovar Typhimurium across a total of 20 fully assembled genomes deposited into GenBank. Cross-annotation resulted in a 32% increase in the number of core genes and a 3-fold decrease in the number of genes identified as mosaic genes (i.e., genes present in some strains only) by the original annotation. Of the remaining noncore genes, the vast majority were prophage genes, and 255 of the nonphage genes were actually of core origin but lost in some strains upon the emergence of the S Typhimurium serovar, suggesting that the chromosomal portion of the S Typhimurium genome acquired a very limited number of novel genes other than prophages. Only horizontally acquired nonphage genes related to bacterial fitness or virulence were found in four recently sequenced isolates, all located on three different genomic islands that harbor multidrug resistance determinants. Thus, the extensive use of antimicrobials could be the main selection force behind the new fitness gene acquisition and the emergence of novel Salmonella pathotypes. IMPORTANCE: Significant discrepancies in the annotations of bacterial genomes could mislead the conclusions about evolutionary origin of chromosomal genes, as we demonstrate here via a cross-annotation-based analysis of Salmonella Typhimurium genomes from GenBank. We conclude that despite being able to infect a broad range of vertebrate hosts, the genomic diversity of S Typhimurium strains is almost exclusively limited to gene loss and the transfer of prophage DNA. Only nonphage chromosomal genes acquired after the emergence of the serovar are linked to the genomic islands harboring multidrug resistance factors. Since the fitness factors could lead to increased virulence, this poses an important research question: could overuse or misuse of antimicrobials act as selection forces for the emergence of more pathogenic strains of Salmonella?

Synonymous and nonsynonymous distances help untangle convergent evolution and recombination.

When estimating a phylogeny from a multiple sequence alignment, researchers often assume the absence of recombination. However, if recombination is present, then tree estimation and all downstream analyses will be impacted, because different segments of the sequence alignment support different phylogenies. Similarly, convergent selective pressures at the molecular level can also lead to phylogenetic tree incongruence across the sequence alignment. Current methods for detection of phylogenetic incongruence are not equipped to distinguish between these two different mechanisms and assume that the incongruence is a result of recombination or other horizontal transfer of genetic information. We propose a new recombination detection method that can make this distinction, based on synonymous codon substitution distances. Although some power is lost by discarding the information contained in the nonsynonymous substitutions, our new method has lower false positive probabilities than the comparable recombination detection method when the phylogenetic incongruence signal is due to convergent evolution. We apply our method to three empirical examples, where we analyze: (1) sequences from a transmission network of the human immunodeficiency virus, (2) tlpB gene sequences from a geographically diverse set of 38 Helicobacter pylori strains, and (3) hepatitis C virus sequences sampled longitudinally from one patient.

Conformational inactivation induces immunogenicity of the receptor-binding pocket of a bacterial adhesin.

Inhibiting antibodies targeting receptor-binding pockets in proteins is a major focus in the development of vaccines and in antibody-based therapeutic strategies. Here, by using a common mannose-specific fimbrial adhesin of Escherichia coli, FimH, we demonstrate that locking the adhesin in a low-binding conformation induces the production of binding pocket-specific, adhesion-inhibiting antibodies. A di-sulfide bridge was introduced into the conformationally dynamic FimH lectin domain, away from the mannose-binding pocket but rendering it defective with regard to mannose binding. Unlike the native, functionally active lectin domain, the functionally defective domain was potent in inducing inhibitory monoclonal antibodies that blocked FimH-mediated bacterial adhesion to epithelial cells and urinary bladder infection in mice. Inhibition of adhesion involved direct competition between the antibodies and mannose for the binding pocket. Binding pocket-specific inhibitory antibodies also were abundant in polyclonal immune serum raised against the functionally defective lectin domain. The monoclonal antibodies elicited against the binding-defective protein bound to the high-affinity conformation of the adhesin more avidly than to the low-affinity form. However, both soluble mannose and blood plasma more strongly inhibited antibody recognition of the high-affinity FimH conformation than the low-affinity form. We propose that in the functionally active conformation the binding-pocket epitopes are shielded from targeted antibody development by ligand masking and that strong immunogenicity of the binding pocket is unblocked when the adhesive domain is in the nonbinding conformation.

PanCoreGen - Profiling, detecting, annotating protein-coding genes in microbial genomes.

A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing the pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen - a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for a species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars - Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study.

Intensity and Mechanisms of Fluoroquinolone Resistance within the H30 and H30Rx Subclones of Escherichia coli Sequence Type 131 Compared with Other Fluoroquinolone-Resistant E. coli.

The recent expansion of the H30 subclone of Escherichia coli sequence type 131 (ST131) and its CTX-M-15-associated H30Rx subset remains unexplained. Although ST131 H30 typically exhibits fluoroquinolone resistance, so do multiple other E. coli lineages that have not expanded similarly. To determine whether H30 isolates have more intense fluoroquinolone resistance than other fluoroquinolone-resistant E. coli isolates and to identify possible mechanisms, we determined the MICs for four fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin, and norfloxacin) among 89 well-characterized, genetically diverse fluoroquinolone-resistant E. coli isolates (48 non-H30 and 41 H30 [23 H30Rx and 18 H30 non-Rx]). We compared the MICs with the H30 and H30Rx status, the presence/number of nonsynonymous mutations in gyrA, parC, and parE, the presence of aac(6')-1b-cr (an aminoglycoside/fluoroquinolone agent-modifying enzyme), and the efflux pump activity (measured as organic solvent tolerance [OST]). Among 1,518 recent E. coli clinical isolates, ST131 H30 predominated clonally, both overall and among the fluoroquinolone-resistant isolates. Among the 89 study isolates, compared with non-H30 isolates, H30 isolates exhibited categorically higher MICs for all four fluoroquinolone agents, higher absolute ciprofloxacin and norfloxacin MICs, more nonsynonymous mutations in gyrA, parC, and parE (specifically gyrA D87N, parC E84V, and parE I529L), and a numerically higher prevalence of (H30Rx-associated) aac(6')-1b-cr but lower OST scores. All putative resistance mechanisms were significantly associated with the MICs [for aac(6')-1b-cr: ciprofloxacin and norfloxacin only]. parC D87N corresponded with ST131 H30 and parE I529L with ST131 generally. Thus, more intense fluoroquinolone resistance may provide ST131 H30, especially H30Rx [if aac(6')-1b-cr positive], with subtle fitness advantages over other fluoroquinolone-resistant E. coli strains. This urges both parsimonious fluoroquinolone use and a search for other fitness-enhancing traits within ST131 H30.

Greater ciprofloxacin tolerance as a possible selectable phenotype underlying the pandemic spread of the H30 subclone of Escherichia coli sequence type 131.

Minimum bactericidal concentrations (MBCs) for ciprofloxacin were significantly higher among 41 members of the H30 subclone within Escherichia coli sequence type 131 than among 48 other fluoroquinolone-resistant E. coli isolates. This MBC difference, which was not explained by ciprofloxacin MICs, gyrA, parC, and parE mutations, the presence of aac(6')-Ib-cr, or organic solvent tolerance (a surrogate for efflux pump activity), conceivably could have promoted the pandemic emergence of the H30 sequence type 131 subclone.