Sequencing and Functional Annotation of Avian Pathogenic Escherichia coli Serogroup O78 Strains Reveal the Evolution of E. coli Lineages Pathogenic for Poultry via Distinct Mechanisms

Avian pathogenic Escherichia coli (APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resembles E. coli causing extraintestinal human diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic E. coli (ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of χ7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of χ7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of E. coli strains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution of E. coli strains adapted to cause avian or human disease via acquisition of distinct virulence genes.     

Role of avian pathogenic Escherichia coli virulence factors in bacterial interaction with chicken heterophils and macrophages

Avian pathogenic Escherichia coli (APEC) cause extraintestinal disease in avian species via respiratory tract infection. Virulence factors associated with APEC include type 1 and P fimbriae, curli, aerobactin, lipopolysaccharide (LPS), K1 capsular antigen, temperature-sensitive hemagglutinin (Tsh), and an uncharacterized pathogen-specific chromosomal region (the 0-min region). The role of these virulence factors in bacterial interaction with phagocytes was investigated by using mutants of three APEC strains, each belonging to one of the most predominant serogroups O1, O2, and O78. Bacterial cell interaction with avian phagocytes was tested with primary cultures of chicken heterophils and macrophages. The presence of type 1 fimbriae and, in contrast, the absence of P fimbriae, K1 capsule, O78 antigen, and the 0-min region promoted bacterial association with chicken heterophils and macrophages. The presence of type 1 and P fimbriae, O78 antigen, and the 0-min region seemed to protect bacteria against the bactericidal effect of phagocytes, especially heterophils. The tested virulence factors seemed to have a limited role in intracellular survival for up to 48 h in macrophages. Generally, opsonized and nonopsonized bacteria were eliminated to the same extent, but in some cases, unopsonized bacteria were eliminated to a greater extent than opsonized bacteria. These results confirm the important role of type 1 fimbriae in promotion of initial phagocytosis, but nevertheless indicate a role for type 1 fimbriae in the protection of bacteria from subsequent killing, at least in heterophils. The results also indicate a role for K1 capsule, O78 antigen, P fimbriae, and the 0-min region in initial avoidance of phagocytosis, but demonstrate an additional role for O78 antigen, P fimbriae, and the 0-min region in subsequent protection against the bactericidal effects of phagocytes after bacterial association has occurred.     

Efficient Prediction of In Vitro Piroxicam Release and Diffusion From Topical Films Based on Biopolymers Using Deep Learning Models and Generative Adversarial Networks

The purpose of this study was to simultaneously predict the drug release and skin permeation of Piroxicam (PX) topical films based on Chitosan (CTS), Xanthan gum (XG) and its Carboxymethyl derivatives (CMXs) as matrix systems. These films were prepared by the solvent casting method, using Tween 80 (T80) as a permeation enhancer. All of the prepared films were assessed for their physicochemical parameters, their in vitro drug release and ex vivo skin permeation studies. Moreover, deep learning models and machine learning models were applied to predict the drug release and permeation rates. The results indicated that all of the films exhibited good consistency and physicochemical properties. Furthermore, it was noticed that when T80 was used in the optimal formulation (F8) based on CTS-CMX3, a satisfactory drug release pattern was found where 99.97% of PX was released and an amount of 1.18 mg/cm² was permeated after 48 h. Moreover, Generative Adversarial Network (GAN) efficiently enhanced the performance of deep learning models and DNN was chosen as the best predictive approach with MSE values equal to 0.00098 and 0.00182 for the drug release and permeation kinetics, respectively. DNN precisely predicted PX dissolution profiles with f₂ values equal to 99.99 for all the formulations.     

Role of virulence factors in resistance of avian pathogenic Escherichia coli to serum and in pathogenicity

In chickens, colibacillosis is caused by avian pathogenic Escherichia coli (APEC) via respiratory tract infection. Many virulence factors, including type 1 (F1A) and P (F11) fimbriae, curli, aerobactin, K1 capsule, and temperature-sensitive hemagglutinin (Tsh) and plasmid DNA regions have been associated with APEC. A strong correlation between serum resistance and virulence has been demonstrated, but roles of virulence factors in serum resistance have not been well elucidated. By using mutants of APEC strains TK3, MT78, and chi7122, which belong to serogroups O1, O2, and O78, respectively, we investigated the role of virulence factors in resistance to serum and pathogenicity in chickens. Our results showed that serum resistance is one of the pathogenicity mechanisms of APEC strains. Virulence factors that increased bacterial resistance to serum and colonization of internal organs of infected chickens were O78 lipopolysaccharide of E. coli chi7122 and the K1 capsule of E. coli MT78. In contrast, curli, type 1, and P fimbriae did not appear to contribute to serum resistance. We also showed that the iss gene, which was previously demonstrated to increase resistance to serum in certain E. coli strains, is located on plasmid pAPEC-1 of E. coli chi7122 but does not play a major role in resistance to serum for strain chi7122.     

Splenic foam cells: a clinico-pathologic analysis of 92 splenectomized patients.

Spleens from 92 patients who underwent splenectomy for various indications were examined. These indications included hematologic disease in 38 patients and nonhematologic problems in 54. Of all the group foam cells were seen in hematoxylin and eosin (HE)-stained sections of spleens from 12 patients. This number increased to 21 (23%) when the diastase periodic acid-Schiff stain was used as a screening test. The cells in these 9 additional cases were too scanty to be observed on HE-stained sections alone. All the 21 spleens with positive foam cells were from patients with hematologic disease, specifically beta-thalassemia major, hemoglobin S/beta-thalassemia, hemoglobin AS, and idiopathic thrombocytopenic purpura. None of the spleens from the 54 nonhematologic patients showed foam cells. Factors that determine the probability of presence of foam cells were calculated. Also, certain differences in the staining reactions of foam cells were observed. The results of both, and the relation of these foam cells to the 'syndrome of the sea-blue histiocyte' are presented and discussed.     

Characterization of the Contribution to Virulence of Three Large Plasmids of Avian Pathogenic Escherichia coli χ7122 (O78:K80:H9)

Despite the fact that the presence of multiple large plasmids is a defining feature of extraintestinal pathogenic Escherichia coli (ExPEC), such as avian pathogenic E. coli (APEC), and despite the fact that these bacteria pose a considerable threat to both human and animal health, characterization of these plasmids is still limited. In this study, after successfully curing APEC of its plasmids, we were able to investigate, for the first time, the contribution to virulence of three plasmids, pAPEC-1 (103 kb), pAPEC-2 (90 kb), and pAPEC-3 (60 kb), from APEC strain χ7122 individually as well as in all combinations in the wild-type background. Characterization of the different strains revealed unique features of APEC virulence. In vivo assays showed that curing the three plasmids resulted in severe attenuation of virulence. The presence of different plasmids and combinations of plasmids resulted in strains with different pathotypes and levels of virulence, reflecting the diversity of APEC strains associated with colibacillosis in chickens. Unexpectedly, our results associated the decrease in growth of some strains in some media with the virulence of APEC, and the mechanism was associated with some combinations of plasmids that included pAPEC-1. This study provided new insights into the roles of large plasmids in the virulence, growth, and evolution of APEC by showing for the first time that both the nature of plasmids and combinations of plasmids have an effect on these phenomena. It also provided a plausible explanation for some of the conflicting results related to the virulence of ExPEC strains. This study should help us understand the virulence of other ExPEC strains and design more efficient infection control strategies.Escherichia coli strains are members of the normal intestinal microflora of most mammals and birds. They colonize their primary habitat, the lower intestinal tract of the host, within the first few hours of the host''s life (37, 54). E. coli strains are very versatile organisms, and the environment is considered their secondary habitat; approximately one-half of all living E. coli cells are actually living outside their hosts. Even though most E. coli strains are commensals and their presence provides a benefit to the host, a subset of these bacteria has acquired the ability to cause intestinal and extraintestinal diseases. These bacteria can be distinguished from commensals by their virulence factors (29, 37).Extraintestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health due to potential economic losses stemming from illness (30, 55, 62). ExPECs are responsible for a broad spectrum of infections in humans, including urinary tract infection (UTI), newborn meningitis (NBM), and septicemia. In addition, they are involved in animal diseases, such as avian colibacillosis, one of the most significant and widespread infectious diseases occurring in poultry and the cause of increased mortality, condemnations, and decreased production (3, 16). The most common disease syndromes associated with E. coli in birds are lower-respiratory-tract infections (air sacculitis), cellulites, meningitis, and septicemia (3).The different groups of E. coli have evolved mainly by acquisition of genes via horizontal gene transfer, a common phenomenon in bacteria that occurs even between very distantly related species (12, 45). This mechanism contributes to the evolution of E. coli variants, resulting in the development of novel strains and pathotypes. Conjugative plasmids are known to mediate transfer of genes between bacteria in diverse environments (42, 67). Acquisition of plasmids by bacteria is one of the fastest ways for survival in and adaptation to one or multiple hosts, as plasmids can encode multiple traits, including antibiotic and heavy metal resistance, virulence, and persistence in different environments (21).ExPEC strains (ExPECs) are differentiated from other pathotypes by the presence of specific virulence genes that allow them to spread systemically in hosts (62). ExPECs, particularly APEC isolates, carry multiple large plasmids (13, 32-35) belonging to different incompatibility groups (35), and the most prevalent plasmids in APEC strains (APECs) are the IncFIB, IncFIC, IncFIIA, IncI1, incP, incB/O, and IncN plasmids, some of which encode virulence factors. Additionally, plasmids encoding multiple drug resistance have been isolated from both APEC and uropathogenic E. coli (UPEC) strains. To date, few studies have undertaken sequencing and characterization of plasmids from avian isolates, particularly the ColV and ColBM plasmids from the IncFIB incompatibility group, which are considered common among ExPEC strains (22, 32, 33, 48, 66). Each of these plasmids has a conserved region harboring the FIB replicon, the ColV and/or ColBM operon, several known virulence genes, and iron acquisition and transport operons. According to recent studies the zoonotic risk seems to be related to the presence of large plasmids in APECs (48, 61).A fuller understanding of ExPEC virulence mechanisms is needed to develop treatments and preventative measures for use against ExPEC infections (55). Reductionism has been used for many years as a critical and powerful tool for identification of key genes responsible for microbial pathogenesis. However, the limitations of this approach for understanding the pathogenicity of bacteria include the multifactorial nature of virulence and the complex cross-regulation of gene expression. The ExPECs that cause diseases in humans and animals are very diverse, and although serotype and virulence factors are related to this diversity, the exact molecular mechanism behind the extensive diversity has not been elucidated yet.APEC strain χ7122 (O78:K80:H9) has been used for many years as a model strain to study the molecular mechanisms of APEC pathogenicity. The results of such studies have contributed greatly to increasing our understanding of the virulence of both human and animal ExPECs. This bacterium has three large plasmids, pAPEC-1 (103 kb), pAPEC-2 (90 kb), and pAPEC-3 (60 kb) (48). Most known virulence factors associated with APEC, including iron acquisition systems, tsh, and colicin V, are located on pAPEC-1, whereas the contents of pAPEC-2 and pAPEC-3 are completely unknown.Despite the fact that the presence of multiple large plasmids is a defining feature of the APEC pathotype (13, 32-35), characterization of these plasmids is still very limited. The exact role of many of them, as well the epistatic interactions between them, are unknown. The study of these plasmids has been complicated by their diversity and by the difficulty of curing them from the wild type. The few previous studies dedicated to understanding the role of the large plasmids of APEC in virulence were done in either E. coli K-12 (15, 31, 63) or avian commensal E. coli backgrounds (61, 70), which did not necessarily show the true functions of these plasmids in the wild-type background host strain.A plasmidless strain obtained from a wild-type APEC strain would provide a better background to evaluate the potential virulence of individual plasmids. In this study, after successfully curing APEC of its plasmids, we were able to investigate the contribution to virulence of each of the three large plasmids of APEC χ7122 by generating a plasmidless strain, strains with each plasmid individually, and strains with two plasmids in different combinations. We then determined the genetic locations of different virulence genes and compared the plasmid-containing derivative strains to the wild-type strain in terms of virulence, growth rate, serum resistance, iron uptake, and lipopolysaccharide (LPS) and iron-regulated outer membrane protein (IROMP) profiles. The results of this study provide new insights into the role of large plasmids in virulence, growth, and evolution of APEC by showing for the first time that both the nature of plasmids and combinations of plasmids have an effect on these factors. They also provide a plausible explanation for some conflicting results related to the virulence of ExPECs.