The Lack of Natural IgM Increases Susceptibility and Impairs Anti-Vi Polysaccharide IgG Responses in a Mouse Model of Typhoid.

Circulating IgM present in the body prior to any apparent Ag exposure is referred to as natural IgM. Natural IgM provides protective immunity against a variety of pathogens. Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever in humans. Because mice are not permissive to S. Typhi infection, we employed a murine model of typhoid using S. enterica serovar Typhimurium expressing the Vi polysaccharide (ViPS) of S. Typhi (S. Typhimurium strain RC60) to evaluate the role of natural IgM in pathogenesis. We found that natural mouse IgM binds to S. Typhi and S. Typhimurium. The severity of S. Typhimurium infection in mice is dependent on presence of the natural resistance-associated macrophage protein 1 (Nramp1) allele; therefore, we infected mice deficient in secreted form of IgM (sIgM) on either a Nramp1-resistant (129S) or -susceptible (C57BL/6J) background. We found that the lack of natural IgM results in a significantly increased susceptibility and an exaggerated liver pathology regardless of the route of infection or the Nramp1 allele. Reconstitution of sIgM-/- mice with normal mouse serum or purified polyclonal IgM restored the resistance to that of sIgM+/+ mice. Furthermore, immunization of sIgM-/- mice with heat-killed S. Typhi induced a significantly reduced anti-ViPS IgG and complement-dependent bactericidal activity against S. Typhi in vitro, compared with that of sIgM+/+ mice. These findings indicate that natural IgM is an important factor in reducing the typhoid severity and inducing an optimal anti-ViPS IgG response to vaccination.

Increased Production of Outer Membrane Vesicles by Salmonella Interferes with Complement-Mediated Innate Immune Attack.

Bacterial outer membrane vesicles (OMVs) enriched with bioactive proteins, toxins, and virulence factors play a critical role in host-pathogen and microbial interactions. The two-component system PhoP-PhoQ (PhoPQ) of Salmonella enterica orchestrates the remodeling of outer membrane lipopolysaccharide (LPS) molecules and concomitantly upregulates OMV production. In this study, we document a novel use of nanoparticle tracking analysis to determine bacterial OMV size and number. Among the PhoPQ-activated genes tested, pagC expression had the most significant effect on the upregulation of OMV production. We provide the first evidence that PhoPQ-mediated upregulation of OMV production contributes to bacterial survival by interfering with complement activation. OMVs protected bacteria in a dose-dependent manner, and bacteria were highly susceptible to complement-mediated killing in their absence. OMVs from bacteria expressing PagC bound to complement component C3b in a dose-dependent manner and inactivated it by recruiting complement inhibitor Factor H. As we also found that Factor H binds to PagC, we propose that PagC interferes with complement-mediated killing of Salmonella in the following two steps: first by engaging Factor H, and second, through the production of PagC-enriched OMVs that divert and inactivate the complement away from the bacteria. Since PhoPQ activation occurs intracellularly, the resultant increase in PagC expression and OMV production is suggested to contribute to the local and systemic spread of Salmonella released from dying host cells that supports the infection of new cells. IMPORTANCE Bacterial outer membrane vesicles (OMVs) mediate critical bacterium-bacterium and host-microbial interactions that influence pathogenesis through multiple mechanisms, including the elicitation of inflammatory responses, delivery of virulence factors, and enhancement of biofilm formation. As such, there is a growing interest in understanding the underlying mechanisms of OMV production. Recent studies have revealed that OMV biogenesis is a finely tuned physiological process that requires structural organization and selective sorting of outer membrane components into the vesicles. In Salmonella, outer membrane remodeling and OMV production are tightly regulated by its PhoPQ system. In this study, we demonstrate that PhoPQ-regulated OMV production plays a significant role in defense against host innate immune attack. PhoPQ-activated PagC expression recruits the complement inhibitor Factor H and degrades the active C3 component of complement. Our results provide valuable insight into the combination of tools and environmental signals that Salmonella employs to evade complement-mediated lysis, thereby suggesting a strong evolutionary adaptation of this facultative intracellular pathogen to protect itself during its extracellular stage in the host.

The Not so Good, the Bad and the Ugly: Differential Bacterial Adhesion and Invasion Mediated by Salmonella PagN Allelic Variants.

While advances in genomic sequencing have highlighted significant strain variability between and within Salmonella serovars, only a few protein variants have been directly related to evolutionary adaptation for survival, such as host specificity or differential virulence. The current study investigated whether allelic variation of the Salmonella adhesin/invasin PagN influences bacterial interaction with their receptors. The Salmonella enterica, subspecies enterica serovar Typhi (S. Typhi) allelic variant of PagN was found to bind significantly better to different enterocytes as well as to the extracellular matrix protein laminin than did the major Salmonella enterica, subspecies enterica serovar Typhimurium (S. Typhimurium) allele. The two alleles differed at amino acid residues 49 and 109 in two of the four predicted PagN surface loops, and residue substitution analysis revealed that a glutamic acid at residue 49 increased the adhesive and invasive properties of S. Typhi PagN. PagN sequence comparisons from 542 Salmonella strains for six representative S. enterica serovars and S. diarizonae further supported the role of glutamic acid at residues 49 and 109 in optimizing adhesion to cells and laminin, as well as for cell invasion. In summary, this study characterized unique residues in allelic variants of a virulence factor that participates in the colonization and invasive properties of different Salmonella stains, subspecies and serovars.

Association of Salmonella virulence factor alleles with intestinal and invasive serovars.

BACKGROUND: The role of Salmonella virulence factor (VF) allelic variation in modulating pathogenesis or host specificity has only been demonstrated in a few cases, mostly through serendipitous findings. Virulence factor (VF) alleles from Salmonella enterica subsp. enterica genomes were compared to identify potential associations with the host-adapted invasive serovars Typhi, Dublin, Choleraesuis, and Gallinarum, and with the broad host-range intestinal serovars Typhimurium, Enteritidis, and Newport. RESULTS: Through a bioinformatics analysis of 500 Salmonella genomes, we have identified allelic variants of 70 VFs, many of which are associated with either one of the four host-adapted invasive Salmonella serovars or one of the three broad host-range intestinal serovars. In addition, associations between specific VF alleles and intra-serovar clusters, sequence types (STs) and/or host-adapted FimH adhesins were identified. Moreover, new allelic VF associations with non-typhoidal S. Enteritidis and S. Typhimurium (NTS) or invasive NTS (iNTS) were detected. CONCLUSIONS: By analogy to the previously shown association of specific FimH adhesin alleles with optimal binding by host adapted Salmonella serovars, lineages or strains, we predict that some of the identified association of other VF alleles with host-adapted serovars, lineages or strains will reflect specific contributions to host adaptation and/or pathogenesis. The identification of these allelic associations will support investigations of the biological impact of VF alleles and better characterize the role of allelic variation in Salmonella pathogenesis. Most relevant functional experiments will test the potential causal contribution of the detected FimH-associated VF variants in host adapted virulence.

Diversified sources for human infections by Salmonella enterica serovar newport.

Salmonella enterica Newport (S. Newport), with phylogenetic diversity feature, contributes to significant public health concerns. Our previous study suggested that S. Newport from multiple animal-borne routes, with distinct antibiotic resistant pattern, might transmit to human. However, their genetic information was lacking. As a complement to the earlier finding, we investigate the relationship between each other among the hosts, sources, genotype and antibiotic resistance in S. Newport. We used the multilocus sequence typing (MLST) in conjunction with minimum inhibitory concentration of 16 antibiotics of globally sampled 1842 S. Newport strains, including 282 newly contributed Chinese strains, to evaluate this association. Our analysis reveals that sequence types (STs) are significantly associated with different host sources, including livestock (ST45), birds (ST5), contaminated water and soil (ST118), reptiles (ST46) and seafood (ST31). Importantly, ST45 contained most of (344/553) the multi-drug resistance (MDR) strains, which were believed to be responsible for human MDR bacterial infections. Chinese isolates were detected to form two unique lineages of avian (ST808 group) and freshwater animal (ST2364 group) origin. Taken together, genotyping information of S. Newport could serve to improve Salmonella source-originated diagnostics and guide better selection of antibiotic therapy against Salmonella infections.

Phylogroup and virulence gene association with clinical characteristics of Escherichia coli urinary tract infections from dogs and cats.

Escherichia coli isolates from infections outside the gastrointestinal tract are termed extra-intestinal pathogenic E. coli (ExPEC) and can be divided into different subpathotypes; one of these is uropathogenic E. coli (UPEC). The frequency with which UPEC strains cause urinary tract infections in dogs and cats is not well documented. We used an oligonucleotide microarray to characterize 60 E. coli isolates associated with the urinary tract of dogs ( n = 45) and cats ( n = 15), collected from 2004 to 2007, into ExPEC and UPEC and to correlate results with patient clinical characteristics. Microarray analysis was performed, and phylogroup was determined by a quadruplex PCR assay. Isolates that were missing 1 or 2 of the gene determinants representative of a function (capsule, iron uptake related genes, or specific adhesins) were designated as "non-classifiable" by microarray. Phylogroup B2 was positively associated with the UPEC subpathotype ( p < 0.0005) and negatively associated with "non-classifiable" isolates ( p < 0.0005). Phylogroup D was positively associated with ExPEC pathotype ( p = 0.025) and negatively associated with UPEC subpathotype ( p = 0.014). The ExPEC pathotype was positively associated with hospitalization for one or more days ( p = 0.031). The UPEC subpathotype was negatively associated with previous antimicrobial therapy ( p = 0.045) and previous hospitalization within the 3 mo prior to the positive culture ( p = 0.041). The UPEC subpathotype was positively associated with prostatitis ( p = 0.073) and negatively associated with current immunosuppressive therapy ( p = 0.090). Our results indicate that the case history observations may be critically important during the interpretation of laboratory results to encourage judicious use of antimicrobials.

Corrigendum: Allelic variation contributes to bacterial host specificity.

This corrects the article DOI: 10.1038/ncomms9754.

Cooperation of Adhesin Alleles in Salmonella-Host Tropism.

Allelic combinations and host specificities for three fimbrial adhesins, FimH, BcfD, and StfH, were compared for 262 strains of Salmonella enterica serovar Newport, a frequent human and livestock pathogen. Like FimH, BcfD had two major alleles (designated A and B), whereas StfH had two allelic groups, each with two alleles (subgroup A1 and A2 and subgroup B1 and B2). The most prevalent combinations of FimH/BcfD/StfH alleles in S. Newport were A/A/A1 and B/B/B1. The former set was most frequently found in bovine and porcine strains, whereas the latter combination was most frequently found in environmental and human isolates. Bacteria genetically engineered to express Fim, Bcf, or Stf fimbriae on their surface were tested with the different alleles for binding to human, porcine, and bovine intestinal epithelial cells. The major allelic combinations with bovine and porcine strains (A/A/A1) or with human isolates (B/B/B1) provided at least two alleles capable of binding significantly better than the other alleles to an intestinal epithelial cell line from the respective host(s). However, each combination of alleles kept at least one allele mediating binding to an intestinal epithelial cell from another host. These findings indicated that allelic variation in multiple adhesins of S. Newport contributes to bacterial adaptation to certain preferential hosts without losing the capacity to maintain a broad host range. IMPORTANCESalmonella enterica remains a leading foodborne bacterial pathogen in the United States; infected livestock serve often as the source of contaminated food products. A study estimated that over a billion Salmonella gastroenteritis cases and up to 33 million typhoid cases occur annually worldwide, with 3.5 million deaths. Although many Salmonella strains with a broad host range present preferential associations with certain host species, it is not clear what determines the various levels of host adaptation. Here, causal properties of host associations were determined with allelic variants of three colonization factors of S. enterica serovar Newport, a most frequent zoonotic serovar. This is the first study that related not only individual but also a small group of host-associated gene variants with functional properties that cooperate to determine the level of host-adapted virulence. The detected associations should help to identify sources of Salmonella infections in both humans and animals.

Animal Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors: adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17, and F18 fimbriae. Once established in the animal small intestine, ETEC produce enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes: heat-labile toxins that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This review describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics, and the identification of potential new targets by genomics are presented in the context of animal ETEC.

Genomic Analysis of Salmonella enterica Serovar Typhimurium Characterizes Strain Diversity for Recent U.S. Salmonellosis Cases and Identifies Mutations Linked to Loss of Fitness under Nitrosative and Oxidative Stress.

UNLABELLED: Salmonella enterica serovar Typhimurium is one of the most common S. enterica serovars associated with U.S. foodborne outbreaks. S. Typhimurium bacteria isolated from humans exhibit wide-ranging virulence phenotypes in inbred mice, leading to speculation that some strains are more virulent in nature. However, it is unclear whether increased virulence in humans is related to organism characteristics or initial treatment failure due to antibiotic resistance. Strain diversity and genetic factors contributing to differential human pathogenicity remain poorly understood. We reconstructed phylogeny, resolved genetic population structure, determined gene content and nucleotide variants, and conducted targeted phenotyping assays for S. Typhimurium strains collected between 1946 and 2012 from humans and animals in the United States and abroad. Strains from recent U.S. salmonellosis cases were associated with five S. Typhimurium lineages distributed within three phylogenetic clades, which are not restricted by geography, year of acquisition, or host. Notably, two U.S. strains and four Mexican strains are more closely related to strains associated with human immunodeficiency virus (HIV)-infected individuals in sub-Saharan Africa than to other North American strains. Phenotyping studies linked variants specific to these strains in hmpA and katE to loss of fitness under nitrosative and oxidative stress, respectively. These results suggest that U.S. salmonellosis is caused by diverse S. Typhimurium strains circulating worldwide. One lineage has mutations in genes affecting fitness related to innate immune system strategies for fighting pathogens and may be adapting to immunocompromised humans by a reduction in virulence capability, possibly due to a lack of selection for its maintenance as a result of the worldwide HIV epidemic. IMPORTANCE: Nontyphoidal Salmonella bacteria cause an estimated 1.2 million illnesses annually in the United States, 80 million globally, due to ingestion of contaminated food or water. Salmonella Typhimurium is one of the most common serovars associated with foodborne illness, causing self-limiting gastroenteritis and, in approximately 5% of infected patients, systemic infection. Although some S. Typhimurium strains are speculated to be more virulent than others, it is unknown how strain diversity and genetic factors contribute to differential human pathogenicity. Ours is the first study to examine the diversity of S. Typhimurium associated with recent cases of U.S. salmonellosis and to provide some initial correlation between observed genotypes and phenotypes. Definition of specific S. Typhimurium lineages based on such phenotype/genotype correlations may identify strains with greater capability of associating with specific food sources, allowing outbreaks to be more quickly identified. Additionally, defining simple correlates of pathogenesis may have predictive value for patient outcome.

Allelic variation contributes to bacterial host specificity.

Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts.

One-Step Identification of Five Prominent Chicken Salmonella Serovars and Biotypes.

Based on bacterial genomic data, we developed a one-step multiplex PCR assay to identify Salmonella and simultaneously differentiate the two invasive avian-adapted S. enterica serovar Gallinarum biotypes Gallinarum and Pullorum, and the most frequent, specific, and asymptomatic colonizers of chickens, serovars Enteritidis, Heidelberg, and Kentucky.

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum.

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.

Adhesive properties of YapV and paralogous autotransporter proteins of Yersinia pestis.

Yersinia pestis is the causative agent of plague. This bacterium evolved from an ancestral enteroinvasive Yersinia pseudotuberculosis strain by gene loss and acquisition of new genes, allowing it to use fleas as transmission vectors. Infection frequently leads to a rapidly lethal outcome in humans, a variety of rodents, and cats. This study focuses on the Y. pestis KIM yapV gene and its product, recognized as an autotransporter protein by its typical sequence, outer membrane localization, and amino-terminal surface exposure. Comparison of Yersinia genomes revealed that DNA encoding YapV or each of three individual paralogous proteins (YapK, YapJ, and YapX) was present as a gene or pseudogene in a strain-specific manner and only in Y. pestis and Y. pseudotuberculosis. YapV acted as an adhesin for alveolar epithelial cells and specific extracellular matrix (ECM) proteins, as shown with recombinant Escherichia coli, Y. pestis, or purified passenger domains. Like YapV, YapK and YapJ demonstrated adhesive properties, suggesting that their previously related in vivo activity is due to their capacity to modulate binding properties of Y. pestis in its hosts, in conjunction with other adhesins. A differential host-specific type of binding to ECM proteins by YapV, YapK, and YapJ suggested that these proteins participate in broadening the host range of Y. pestis. A phylogenic tree including 36 Y. pestis strains highlighted an association between the gene profile for the four paralogous proteins and the geographic location of the corresponding isolated strains, suggesting an evolutionary adaption of Y. pestis to specific local animal hosts or reservoirs.

Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella.

BACKGROUND: Autotransporter proteins represent a treasure trove for molecular engineers who modify Gram-negative bacteria for the export or secretion of foreign proteins across two membrane barriers. A particularly promising direction is the development of autotransporters as antigen display or secretion systems. Immunologists have been using ovalbumin as a reporter antigen for years and have developed sophisticated tools to detect specific T cells that respond to ovalbumin. Although ovalbumin-expressing bacteria are being used to trace T cell responses to colonizing or invading pathogens, current constructs for ovalbumin presentation have not been optimized. RESULTS: The activation of T helper cells in response to ovalbumin was improved by displaying the OVA-CD4 reporter epitope as a multimer on the surface of Salmonella and fused to the autotransporter MisL. Expression was optimized by including tandem in vivo promoters and two post-segregational killing systems for plasmid stabilization. CONCLUSIONS: The use of an autotransporter protein to present relevant epitope repeats on the surface of bacteria, combined with additional techniques favoring stable and efficient in vivo transcription, optimizes antigen presentation to T cells. The technique of multimeric epitope surface display should also benefit the development of new Salmonella or other enterobacterial vaccines.

Allelic variation in Salmonella: an underappreciated driver of adaptation and virulence.

Salmonella enterica causes substantial morbidity and mortality in humans and animals. Infection and intestinal colonization by S. enterica require virulence factors that mediate bacterial binding and invasion of enterocytes and innate immune cells. Some S. enterica colonization factors and their alleles are host restricted, suggesting a potential role in regulation of host specificity. Recent data also suggest that colonization factors promote horizontal gene transfer of antimicrobial resistance genes by increasing the local density of Salmonella in colonized intestines. Although a profusion of genes are involved in Salmonella pathogenesis, the relative importance of their allelic variation has only been studied intensely in the type 1 fimbrial adhesin FimH. Although other Salmonella virulence factors demonstrate allelic variation, their association with specific metadata (e.g., host species, disease or carrier state, time and geographic place of isolation, antibiotic resistance profile, etc.) remains to be interrogated. To date, genome-wide association studies (GWAS) in bacteriology have been limited by the paucity of relevant metadata. In addition, due to the many variables amid metadata categories, a very large number of strains must be assessed to attain statistically significant results. However, targeted approaches in which genes of interest (e.g., virulence factors) are specifically sequenced alleviates the time-consuming and costly statistical GWAS analysis and increases statistical power, as larger numbers of strains can be screened for non-synonymous single nucleotide polymorphisms (SNPs) that are associated with available metadata. Congruence of specific allelic variants with specific metadata from strains that have a relevant clinical and epidemiological history will help to prioritize functional wet-lab and animal studies aimed at determining cause-effect relationships. Such an approach should be applicable to other pathogens that are being collected in well-curated repositories.

Structural basis for the specific recognition of dual receptors by the homopolymeric pH 6 antigen (Psa) fimbriae of Yersinia pestis.

The pH 6 antigen (Psa) of Yersinia pestis consists of fimbriae that bind to two receptors: ß1-linked galactosyl residues in glycosphingolipids and the phosphocholine group in phospholipids. Despite the ubiquitous presence of either moiety on the surface of many mammalian cells, Y. pestis appears to prefer interacting with certain types of human cells, such as macrophages and alveolar epithelial cells of the lung. The molecular mechanism of this apparent selectivity is not clear. Site-directed mutagenesis of the consensus choline-binding motif in the sequence of PsaA, the subunit of the Psa fimbrial homopolymer, identified residues that abolish galactosylceramide binding, phosphatidylcholine binding, or both. The crystal structure of PsaA in complex with both galactose and phosphocholine reveals separate receptor binding sites that share a common structural motif, thus suggesting a potential interaction between the two sites. Mutagenesis of this shared structural motif identified Tyr126, which is part of the choline-binding consensus sequence but is found in direct contact with the galactose in the structure of PsaA, important for both receptor binding. Thus, this structure depicts a fimbrial subunit that forms a polymeric adhesin with a unique arrangement of dual receptor binding sites. These findings move the field forward by providing insights into unique types of multiple receptor-ligand interactions and should steer research into the synthesis of dual receptor inhibitor molecules to slow down the rapid progression of plague.

Crystallization and preliminary X-ray diffraction analysis of PsaA, the adhesive pilin subunit that forms the pH 6 antigen on the surface of Yersinia pestis.

Yersinia pestis has been responsible for a number of high-mortality epidemics throughout human history. Like all other bacterial infections, the pathogenesis of Y. pestis begins with the attachment of bacteria to the surface of host cells. At least five surface proteins from Y. pestis have been shown to interact with host cells. Psa, the pH 6 antigen, is one of them and is deployed on the surface of bacteria as thin flexible fibrils that are the result of the polymerization of a single PsaA pilin subunit. Here, the crystallization of recombinant donor-strand complemented PsaA by the hanging-drop vapor-diffusion method is reported. X-ray diffraction data sets were collected to 1.9 Šresolution from a native crystal and to 1.5 Šresolution from a bromide-derivatized crystal. These crystals displayed the symmetry of the orthorhombic space group P222(1), with unit-cell parameters a = 26.3, b = 54.6, c = 102.1 Å. Initial phases were derived from single isomorphous replacement with anomalous scattering experiments, resulting in an electron-density map that showed a single molecule in the crystallographic asymmetric unit. Sequence assignment was aided by residues binding to bromide ions of the heavy-atom derivative.

Microfluidic PCR combined with pyrosequencing for identification of allelic variants with phenotypic associations among targeted Salmonella genes.

A novel targeted massive parallel sequencing approach identified genetic variation in eight known or predicted fimbrial adhesins for 46 Salmonella strains. The results highlight associations between specific adhesin alleles, host species, and antimicrobial resistance. The differentiation of allelic variants has potential applications for diagnostic microbiology and epidemiological investigations.