Genomic Characterization of a Plasmid-Free and Highly Drug-Resistant Salmonella enterica Serovar Indiana Isolate in China.

The emergence of multi-drug resistant (MDR) Salmonella enterica serovar Indiana (S. Indiana) strains in China is commonly associated with the presence of one or more resistance plasmids harboring integrons pivotal in acquiring antimicrobial resistance (AMR). This study aims to elucidate the genetic makeup of this plasmid-free, highly drug-resistant S. Indiana S1467 strain. Genomic sequencing was performed using Illumina HiSeq 2500 sequencer and PacBio RS II System. Prodigal software predicted putative protein-coding sequences while BLASTP analysis was conducted. The S1467 genome comprises a circular 4,998,300 bp chromosome with an average GC content of 51.81%, encompassing 4709 open reading frames (ORFs). Fifty-four AMR genes were identified, conferring resistance across 16 AMR categories, aligning closely with the strain's antibiotic susceptibility profile. Genomic island prediction unveiled an approximately 51 kb genomic island housing a unique YeeVU toxin-antitoxin system (TAS), a rarity in Salmonella species. This suggests that the AMR gene cluster on the S1467 genomic island may stem from the integration of plasmids originating from other Enterobacteriaceae. This study contributes not only to the understanding of the genomic characteristics of a plasmid-free, highly drug-resistant S. Indiana strain but also sheds light on the intricate mechanisms underlying antimicrobial resistance. The implications of our findings extend to the broader context of horizontal gene transfer between bacterial species, emphasizing the need for continued surveillance and research to address the evolving challenges posed by drug-resistant pathogens.

Exploring the inhibition of the soluble lytic transglycosylase Cj0843c of Campylobacter jejuni via targeting different sites with different scaffolds.

Bacterial lytic transglycosylases (LTs) contribute to peptidoglycan cell wall metabolism and are potential drug targets to potentiate ß-lactam antibiotics to overcome antibiotic resistance. Since LT inhibitor development is underexplored, we probed 15 N-acetyl-containing heterocycles in a structure-guided fashion for their ability to inhibit and bind to the Campylobacter jejuni LT Cj0843c. Ten GlcNAc analogs were synthesized with substitutions at the C1 position, with two having an additional modification at the C4 or C6 position. Most of the compounds showed weak inhibition of Cj0843c activity. Compounds with alterations at the C4 position, replacing the -OH with a -NH2 , and C6 position, the addition of a -CH3 , yielded improved inhibitory efficacy. All 10 GlcNAc analogs were crystallographically analyzed via soaking experiments using Cj0843c crystals and found to bind to the +1 +2 saccharide subsites with one of them additionally binding to the -2 -1 subsite region. We also probed other N-acetyl-containing heterocycles and found that sialidase inhibitors N-acetyl-2,3-dehydro-2-deoxyneuraminic acid and siastatin B inhibited Cj0843c weakly and crystallographically bound to the -2 -1 subsites. Analogs of the former also showed inhibition and crystallographic binding and included zanamivir amine. This latter set of heterocycles positioned their N-acetyl group in the -2 subsite with additional moieties interacting in the -1 subsite. Overall, these results could provide novel opportunities for LT inhibition via exploring different subsites and novel scaffolds. The results also increased our mechanistic understanding of Cj0843c regarding peptidoglycan GlcNAc subsite binding preferences and ligand-dependent modulation of the protonation state of the catalytic E390.

Characterization of the mechanism of bile salt hydrolase substrate specificity by experimental and computational analyses.

Bile salt hydrolases (BSHs) are currently being investigated as target enzymes for metabolic regulators in humans and as growth promoters in farm animals. Understanding structural features underlying substrate specificity is necessary for inhibitor design. Here, we used a multidisciplinary workflow including mass spectrometry, mutagenesis, molecular dynamic simulations, machine learning, and crystallography to demonstrate substrate specificity in Lactobacillus salivarius BSH, the most abundant enzyme in human and farm animal intestines. We show the preference of substrates with a taurine head and a dehydroxylated sterol ring for hydrolysis. A regression model that correlates the relative rates of hydrolysis of various substrates in various enzyme mutants with the residue-substrate interaction energies guided the identification of structural determinants of substrate binding and specificity. In addition, we found T208 from another BSH protomer regulating the hydrolysis. The designed workflow can be used for fast and comprehensive characterization of enzymes with a broad range of substrates.

Critical role of the RpoE stress response pathway in polymyxin resistance of Escherichia coli.

OBJECTIVES: Polymyxins, including colistin, are the drugs of last resort to treat MDR bacterial infections in humans. In-depth understanding of the molecular basis and regulation of polymyxin resistance would provide new therapeutic opportunities to combat increasing polymyxin resistance. Here we aimed to identify novel targets that are crucial for polymyxin resistance using Escherichia coli BL21(DE3), a unique colistin-resistant model strain. METHODS: BL21(DE3) was subjected to random transposon mutagenesis for screening colistin-susceptible mutants. The insertion sites of desired mutants were mapped; the key genes of interest were also inactivated in different strains to examine functional conservation. Specific genes in the known PmrAB and PhoPQ regulatory network were inactivated to examine crosstalk among different pathways. Lipid A species and membrane phospholipids were analysed by normal phase LC/MS. RESULTS: Among eight mutants with increased susceptibility to colistin, five mutants contained different mutations in three genes (rseP, degS and surA) that belong to the RpoE stress response pathway. Inactivation of rpoE, pmrB, eptA or pmrD led to significantly increased susceptibility to colistin; however, inactivation of phoQ or eptB did not change colistin MIC. RpoE mutation in different E. coli and Salmonella resistant strains all led to significant reduction in colistin MIC (16-32-fold). Inactivation of rpoE did not change the lipid A profile but significantly altered the phospholipid profile. CONCLUSIONS: Inactivation of the important members of the RpoE regulon in polymyxin-resistant strains led to a drastic reduction in polymyxin MIC and an increase of lysophospholipids with no change in lipid A modifications.

Longitudinal surveillance and comparative characterization of Escherichia albertii in wild raccoons in the United States.

Escherichia albertii is an emerging enteric bacterial pathogen causing watery diarrhea, abdominal distension, vomiting and fever in humans. E. albertii has caused many foodborne outbreaks in Japan and was also reported in other countries worldwide. However, the important animal reservoirs of this pathogen are still largely unknown, impeding us to combat this emerging pathogen. Recently, we reported that wild raccoons (Procyon lotor) and broiler chickens are significant reservoirs of E. albertii in Japan and the U.S., respectively. Here, we performed a longitudinal surveillance to monitor prevalence of E. albertii in wild raccoons in the U.S. and conducted comprehensive comparative analyses of the E. albertii of different origins. A total of 289 fecal swab samples were collected from wild raccoons in Tennessee and Kentucky in the U.S. (2018-2020). Approximately 26% (74/289) of the raccoons examined were PCR-positive for E. albertii and eventually 22 E. albertii isolates were obtained. PFGE analysis showed the U.S. raccoon E. albertii were phylogenetically distant even though the corresponding raccoons were captured from a small area. Unlike the high prevalence of multidrug resistance (83%) observed in previous chicken E. albertii survey, antibiotic resistance was rarely observed in all the U.S. raccoon and 22 Japan raccoon strains with only one Japan strain displaying multidrug resistance (2%). Whole genome sequencing of 54 diverse E. albertii strains and subsequent comparative genomics analysis revealed unique clusters that displayed close evolutionary relationships and similar virulence gene profiles among the strains of different origins in terms of geographical locations (e.g., U.S. and Japan) and hosts (raccoon, chicken, swine, and human). Challenge experiment demonstrated raccoon E. albertii strains could successfully colonize in the chicken intestine at 3 and 8 days postinfection. A pilot environmental survey further showed all the four tested water samples from Tennessee river were E. albertii-positive; two different E. albertii strains, isolated from a single water sample, showed close relationships to those of human origin. Together, the findings from this study provide new insights into the ecology, evolution, and pathobiology of E. albertii, and underscore the need to control the emerging E. albertii in a complex ecosystem using One Health approach.

Monoclonal antibody-based indirect competitive ELISA for quantitative detection of Enterobacteriaceae siderophore enterobactin.

Enterobactin (Ent) is a promising indicator to monitor intestinal level of Enterobacteriaceae for assessment of gut inflammation. In this study, we developed a monoclonal antibody (mAb)-based ELISA for Ent quantification. We immunized mice with an Ent conjugate vaccine. An mAb named 2E4, with the highest anti-Ent antibody titer, was selected for developing indirect competitive ELISA (ic-ELISA). The purified mAb 2E4 showed high affinity (3.1 × 10-10 M) and specificity to Ent. The limit of detection of ic-ELISA was 0.39 µg/mL. The intra- and inter-assay recovery rates of standard curve were up to 94.6% with the coefficients of variation between 4.0% and 12.3%, indicating high accuracy, repeatability, and reproducibility of the ic-ELISA. In addition, the ic-ELISA was able to quantitatively detect Ent produced in different bacterial cultures. Collectively, this study developed an ic-ELISA with excellent performance in Ent quantification, laying a solid foundation for Ent-based diagnostics of gut health.

Isolation and characterization of Escherichia albertii originated from the broiler farms in Mississippi and Alabama.

Escherichia albertii is an emerging foodborne enteropathogen with increasing outbreaks worldwide, particularly in Japan recently. However, major features of this zoonotic pathogen, such as prevalence, virulence, and antibiotic resistance (AR), still remain under characterized. In a recent pilot study, we reported isolation of E. albertii from a chicken farm in Tennessee, suggesting chicken is an important reservoir for E. albertii. In this large-scale study, we examined prevalence of E. albertii in 9 farms in Mississippi and Alabama. Of a total of 270 cloacal swabs (30 per farm), 43 were PCR positive and 12 E. albertii strains were isolated with different isolation rates in individual farms ranging from 0 to 23.3 %. Both PFGE and whole genome analysis showed the E. albertii from different farms were phylogenetically distant, but those from the same farm displayed clonal relationships. Consistently, the antibiogram, AR gene profiles, and plasmid replicon types were similar across the strains in the same farm. Notably, 9 of the 12 E. albertii strains displayed multidrug resistance; one strain was even resistant to imipenem, a clinically important carbapenem antibiotic. In addition, comparative genomics analysis showed that two chicken E. albertii clusters displayed very close evolutionary relationships and similar virulence gene profiles to human E. albertii strains. In vitro growth assay demonstrated that the anti-enterobactin antibodies could dramatically inhibit the growth of two representative chicken E. albertii, supporting the feasibility of the novel enterobactin-based immune intervention for controlling this emerging pathogen. Taken together, the findings from this study further indicated chickens as an important reservoir for E. albertii in the U.S., highlighting the need to prevent and control E. albertii in poultry production.

Ex Vivo Evaluation of Egg Yolk IgY Degradation in Chicken Gastrointestinal Tract.

Egg yolk antibody (immunoglobulin Y, IgY), due to its unique features (e.g., cost-effectiveness for mass production), is emerging as a promising passive immune agent and alternative to antibiotics to combat infectious diseases, particularly in livestock. Oral administration of egg yolk IgY is the most common and convenient route that has been extensively investigated for controlling enteric pathogens. However, the in vivo stability of egg yolk IgY in the gastrointestinal (GI) tract, a critical issue for the success of this approach, still has not been clearly elucidated. Our recent study showed instability of orally administered egg yolk IgY in chicken GI tract, as demonstrated by both in vivo and ex vivo evidence. To better understand the magnitude and dynamics of instability of egg yolk IgY in vivo, in this study, we conducted comprehensive ex vivo analyses by spiking hyperimmune egg yolk IgY in fresh GI contents collected from five broilers at each sampling age (2, 4, or 6 weeks). The pH in gizzard slightly increased with age from 2.4 to 3.0, while the pH in the small intestine was around 5.8. ELISA analysis indicated that a short time of treatment (30 or 60 min) of IgY with the gizzard contents from the chickens at 2, 4, and 6 weeks of age greatly reduced specific IgY titer by over 8, 6, and 5 log2 units, respectively, when compared with saline control. However, small intestine content only had a mild effect on egg yolk IgY, leading to 1 log2 unit of reduction in IgY titer upon 30 min of treatment. Consistent with these findings, SDS-PAGE and immunoblotting analyses provided direct evidence demonstrating that egg yolk IgY could be drastically degraded to undetectable level in gizzard content upon as short as 5 min of treatment; however, the IgY was only slightly degraded in small intestine content. Immunoblotting also showed that treatment of IgY with HCl (pH 3.0) for 60 min did not affect its integrity at all, further supporting the enzymatic degradation of IgY in gizzard. Collectively, egg yolk IgY could be substantially degraded in chicken gizzard, highly warranting the development of effective approaches, such as encapsulation, for the controlled release and protection of orally administered egg yolk IgY in livestock.

Campylobacter jejuni genotypes are associated with post-infection irritable bowel syndrome in humans.

Campylobacter enterocolitis may lead to post-infection irritable bowel syndrome (PI-IBS) and while some C. jejuni strains are more likely than others to cause human disease, genomic and virulence characteristics promoting PI-IBS development remain uncharacterized. We combined pangenome-wide association studies and phenotypic assays to compare C. jejuni isolates from patients who developed PI-IBS with those who did not. We show that variation in bacterial stress response (Cj0145_phoX), adhesion protein (Cj0628_CapA), and core biosynthetic pathway genes (biotin: Cj0308_bioD; purine: Cj0514_purQ; isoprenoid: Cj0894c_ispH) were associated with PI-IBS development. In vitro assays demonstrated greater adhesion, invasion, IL-8 and TNFα secretion on colonocytes with PI-IBS compared to PI-no-IBS strains. A risk-score for PI-IBS development was generated using 22 genomic markers, four of which were from Cj1631c, a putative heme oxidase gene linked to virulence. Our finding that specific Campylobacter genotypes confer greater in vitro virulence and increased risk of PI-IBS has potential to improve understanding of the complex host-pathogen interactions underlying this condition.

Passive Immunization of Chickens with Anti-Enterobactin Egg Yolk Powder for Campylobacter Control.

Enterobactin (Ent) is a highly conserved and important siderophore for the growth of many Gram-negative bacterial pathogens. Therefore, targeting Ent for developing innovative intervention strategies has attracted substantial research interest in recent years. Recently, we developed a novel Ent conjugate vaccine that has been demonstrated to be effective for controlling Gram-negative pathogens using both in vitro and in vivosystems. In particular, active immunization of chickens with the Ent conjugate vaccine elicited strong immune responses and significantly reduced intestinal colonization of Campylobacter jejuni, the leading foodborne bacterial pathogen. Given that hyperimmune egg yolk immunoglobulin Y (IgY) has been increasingly recognized as a promising and practical non-antibiotic approach for passive immune protection against pathogens in livestock, in this study, we assessed the efficacy of oral administration of broiler chickens with the anti-Ent hyperimmune egg yolk powder to control C. jejuni colonization in the intestine. However, supplementation of feed with 2% (w/w) of anti-Ent egg yolk powder failed to reduce C. jejuni colonization when compared to the control group. Consistent with this finding, the ELISA titers of the specific IgY in cecum, ileum, duodenum, gizzard, and serum contents were similar between the two groups throughout the trial. Chicken intestinal microbiota also did not change in response to the egg yolk powder treatment. Subsequently, to examine ex vivo stability of the egg yolk IgY, the chicken gizzard and duodenum contents from two independent sources were spiked with the egg yolk antibodies, incubated at 42 °C for different lengths of time, and subjected to ELISA analysis. The specific IgY titers were dramatically decreased in gizzard contents (up to 2048-fold) but were not changed in duodenum contents. Collectively, oral administration of broiler chickens with the anti-Ent egg yolk powder failed to confer protection against intestinal colonization of C. jejuni, which was due to instability of the IgY in gizzard contents as demonstrated by both in vivo and ex vivo evidence.

Critical Role of 3'-Downstream Region of pmrB in Polymyxin Resistance in Escherichia coli BL21(DE3).

Polymyxins, such as colistin and polymyxin B, are the drugs used as a last resort to treat multidrug-resistant Gram-negative bacterial infections in humans. Increasing colistin resistance has posed a serious threat to human health, warranting in-depth mechanistic research. In this study, using a functional cloning approach, we examined the molecular basis of colistin resistance in Escherichia coli BL21(DE3). Five transformants with inserts ranging from 3.8 to 10.7 kb displayed significantly increased colistin resistance, three of which containing pmrB locus and two containing pmrD locus. Stepwise subcloning indicated that both the pmrB with a single G361A mutation and at least a 103 bp downstream region of pmrB are essential for conferring colistin resistance. Analysis of the mRNA level and stability showed that the length of the downstream region drastically affected the pmrB mRNA level but not its half-life. Lipid A analysis, by mass spectrometry, revealed that the constructs containing pmrB with a longer downstream region (103 or 126 bp) have charge-altering l-4-aminoarabinose (Ara4N) and phosphoethanolamine (pEtN) modifications in lipid A, which were not observed in both vector control and the construct containing pmrB with an 86 bp downstream region. Together, the findings from this study indicate that the 3'-downstream region of pmrB is critical for the PmrB-mediated lipid A modifications and colistin resistance in E. coli BL21(DE3), suggesting a novel regulatory mechanism of PmrB-mediated colistin resistance in E. coli.

Evaluation of the Immunogenic Response of a Novel Enterobactin Conjugate Vaccine in Chickens for the Production of Enterobactin-Specific Egg Yolk Antibodies.

Passive immunization with specific egg yolk antibodies (immunoglobulin Y, IgY) is emerging as a promising alternative to antibiotics to control bacterial infections. Recently, we developed a novel conjugate vaccine that could trigger a strong immune response in rabbits directed against enterobactin (Ent), a highly conserved siderophore molecule utilized by different Gram-negative pathogens. However, induction of Ent-specific antibodies appeared to be affected by the choice of animal host and vaccination regimen. It is still unknown if the Ent conjugate vaccine can trigger a specific immune response in layers for the purpose of production of anti-Ent egg yolk IgY. In this study, three chicken vaccination trials with different regimens were performed to determine conditions for efficient production of anti-Ent egg yolk IgY. Purified Ent was conjugated to three carrier proteins, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and CmeC (a subunit vaccine candidate), respectively. Intramuscular immunization of Barred Rock layers with KLH-Ent conjugate four times induced strong immune response against whole conjugate vaccine but the titer of Ent-specific IgY did not change in yolk with only a 4 fold increase detected in serum. In the second trial, three different Ent conjugate vaccines were evaluated in Rhode Island Red pullets with four subcutaneous injections. The KLH-Ent or CmeC-Ent conjugate consistently induced high level of Ent-specific IgY in both serum (up to 2,048 fold) and yolk (up to 1,024 fold) in each individual chicken. However, the Ent-specific immune response was only temporarily and moderately induced using a BSA-Ent vaccination. In the third trial, ten White Leghorn layers were subcutaneously immunized three times with KLH-Ent, leading to consistent and strong immune response against both whole conjugate and the Ent molecule in each chicken; the mean titer of Ent-specific IgY increased approximately 32 and 256 fold in serum and yolk, respectively. Consistent with its potent binding to various Ent derivatives, the Ent-specific egg yolk IgY also inhibited in vitro growth of a representative Escherichia coli strain. Together, this study demonstrated that the novel Ent conjugate vaccine could induce strong, specific, and robust immune response in chickens. The Ent-specific hyperimmune egg yolk IgY has potential for passive immune intervention against Gram-negative infections.

The Transcriptomic and Bioinformatic Characterizations of Iron Acquisition and Heme Utilization in Avibacterium paragallinarum in Response to Iron-Starvation.

Avibacterium paragallinarum is the pathogen of infectious coryza, which is a highly contagious respiratory disease of chickens that brings a potentially serious threat to poultry husbandry. Iron is an important nutrient for bacteria and can be obtained from surroundings such as siderophores and hemophores. To date, the mechanisms of iron acquisition and heme utilization as well as detailed regulation in A. paragallinarum have been poorly understood. In this study, we investigated the transcriptomic profiles in detail and the changes of transcriptomes induced by iron restriction in A. paragallinarum using RNA-seq. Compared with the iron-sufficiency control group, many more differentially expressed genes (DEGs) and cellular functions as well as signaling pathways were verified in the iron-restriction group. Among these DEGs, the majority of genes showed decreased expression and some were found to be uniquely present in the iron-restriction group. With an in-depth study of bioinformatic analyses, we demonstrated the crucial roles of the Hut protein and DUF domain-containing proteins, which were preferentially activated in bacteria following iron restriction and contributed to the iron acquisition and heme utilization. Consequently, RT-qPCR results further verified the iron-related DEGs and were consistent with the RNA-seq data. In addition, several novel sRNAs were present in A. paragallinarum and had potential regulatory roles in iron homeostasis, especially in the regulation of Fic protein to ensure stable expression. This is the first report of the molecular mechanism of iron acquisition and heme utilization in A. paragallinarum from the perspective of transcriptomic profiles. The study will contribute to a better understanding of the transcriptomic response of A. paragallinarum to iron starvation and also provide novel insight into the development of new antigens for potential vaccines against infectious coryza by focusing on these iron-related genes.

Turnover Chemistry and Structural Characterization of the Cj0843c Lytic Transglycosylase of Campylobacter jejuni.

The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell-wall peptidoglycan (PG). Its nonhydrolytic activity sustains cell-wall remodeling and repair. We report herein our structure-function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exolytic and endolytic activities and forms the N-acetyl-1,6-anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference for substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X-ray crystallography and by molecular dynamics simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di- and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.

Isolation and characterization of Escherichia albertii in poultry at the pre-harvest level.

Escherichia albertii, often misidentified as Escherichia coli, has become an emerging foodborne human enteric pathogen. However, the prevalence and major animal reservoirs of this significant pathogen are still not clear. Here, we performed comprehensive microbiological, molecular, comparative genomics and animal studies to understand the status and features of E. albertii in the US domestic and food animals. Although no E. albertii was identified in a total of 1,022 diverse E. coli strains isolated from pets and food animals in a retrospective screening, in a pilot study, E. albertii was successfully isolated from a broiler farm (6 out of 20 chickens). The chicken E. albertii isolates showed clonal relationship as indicated by both pulsed-field gel electrophoresis (PFGE) and whole-genome sequence analysis. The isolated chicken E. albertii displayed multidrug resistance; all the resistance determinants including the extended-spectrum beta-lactamase gene, carried by plasmids, could be conjugatively transferred to E. coli, which was further confirmed by S1-PFGE and Southern hybridization. Whole-genome sequence-based phylogenetic analysis showed the chicken E. albertii strains were phylogenetically close to those of human origins. Challenge experiment demonstrated that the E. albertii strains isolated from human and wild bird could successfully colonize in the chicken intestine. Together, this study, for the first time, reported the isolation of E. albertii in poultry at the pre-hrvest level. The findings from multi-tier characterization of the chicken E. albertii strains indicated the importance of chickens as a reservoir for E. albertii. A large scale of E. albertii survey in poultry production at the pre-harvest level is highly warranted in the future.

Immunization of Chickens with the Enterobactin Conjugate Vaccine Reduced Campylobacter jejuni Colonization in the Intestine.

Campylobacter jejuni is the leading bacterial cause of human enteritis in developed countries. Chicken is the major animal reservoir of C. jejuni and a powerful infection model for human campylobacteriosis. No commercial vaccine against C. jejuni is available to date. The high affinity iron acquisition mediated through enterobactin (Ent), a small siderophore, plays a critical role in the colonization of C. jejuni in the intestine. Recently, an innovative Ent conjugate vaccine has been demonstrated to induce high-level of Ent-specific antibodies in rabbits; the Ent-specific antibodies displayed potent binding ability to Ent and inhibited Ent-dependent growth of C. jejuni. In this study, using specific-pathogen-free (SPF) chickens, we performed three trials to evaluate the immunogenicity of the Ent conjugate vaccine and its efficacy to control C. jejuni colonization in the intestine. The purified Ent was conjugated to the carrier keyhole limpet hemocyanin (KLH). Intramuscular immunization of chickens with the Ent-KLH conjugate for up to three times did not affect the body weight gain, the development of major immune organs and the gut microbiota. In the first two trials, immunizations of chickens with different regimens (two or three times of vaccination) consistently induced strong Ent-specific immune response when compared to control group. Consistent with the high-level of systemic anti-Ent IgG, C. jejuni colonization was significantly reduced by 3-4 log10 units in the cecum in two independent vaccination trials. The third trial demonstrated that single Ent-KLH vaccination is sufficient to elicit high level of systemic Ent-specific antibodies, which could persist for up to eight weeks in chickens. Taken together, the Ent-KLH conjugate vaccine could induce high-level of Ent-specific antibodies in chickens and confer host protection against C. jejuni colonization, which provides a novel strategy for Campylobacter control in poultry and humans.

Enterobactin-specific antibodies inhibit in vitro growth of different gram-negative bacterial pathogens.

Enterobactin (Ent)-mediated high affinity iron acquisition is critically important for Gram-negative bacterial pathogens to survive and infect the host. Recently, we reported an efficient method to prepare novel Ent conjugate vaccines for inducing high level of Ent-specific antibodies, which displayed similar bacteriostatic feature as lipocalins, the host innate immune effectors with potent Ent-binding ability. The Ent-specific antibodies also showed a significant advantage over lipocalins by cross-reacting to various Ent derivatives including salmochelins, the glycosylated Ent that can help enteric pathogens evade the siderophore sequestration by host lipocalins. To demonstrate significant potential of the Ent conjugate vaccine for broader applications to prevent and control various Gram-negative infections in human and animal, in this study, we examined inhibitory effect of Ent-specific antibodies on the in vitro growth of three significant Gram-negative pathogens: Escherichia coli (n = 27), Salmonella enterica (n = 8), and Campylobacter spp. (n = 6). The tested strains were diverse with respect to hosts, geographical origins, serotypes, infection sites and siderophore productions. The Ent-specific antibodies significantly suppressed the growth of each tested strain under iron-restricted conditions. For example, the Ent-specific antibodies consistently exerted 2-5 log10 units of growth reduction on most tested avian pathogenic E. coli (9 of 10 strains) isolated in five countries. Despite various dynamic growth responses observed, notably, the Ent-specific antibodies displayed significantly higher magnitude of growth reduction than lipocalin-2 (up to 5 log10 units of difference) on majority of tested E. coli and S. enterica, which is likely due to sequestration of other siderophores (e.g., salmochelins) by the Ent-specific antibodies. Production of a variety of major siderophores by the tested E. coli and S. enterica strains was examined and confirmed by ultra high performance liquid chromatography-high resolution mass spectrometry analysis. Collectively, this study provides critical and compelling in vitro evidence supporting the feasibility of Ent-based immune interventions against several Gram-negative pathogens.

Evaluation of in ovo vaccination of DNA vaccines for Campylobacter control in broiler chickens.

Campylobacter is the leading bacterial cause of human enteritis in developed countries. Chicken is a major natural host of Campylobacter. Thus, on-farm control of Campylobacter load in poultry would reduce the risk of human exposure to this pathogen. Vaccination is an attractive intervention measure to mitigate Campylobacter in poultry. Our previous studies have demonstrated that Campylobacter outer membrane proteins CmeC (a component of multidrug efflux pump) and CfrA (ferric enterobactin receptor) are feasible and promising candidates for vaccine development. In this study, by targeting these two attractive vaccine candidates, we explored and evaluated a new vaccination strategy, which combines the in ovo vaccination route and novel DNA vaccine formulation, for Campylobacter control in broilers. We observed that direct cloning of cfrA or cmeC gene into the eukaryotic expression vector pCAGGS did not lead to sufficient level of production of the target proteins in the eukaryotic HEK-293 cell line. However, introduction of the Kozak consensus sequence (ACCATGG) in the cloned bacterial genes greatly enhanced production of inserted gene in eukaryotic cells, creating desired DNA vaccines. Subsequently, the validated DNA vaccines were prepared and used for two independent in ovo vaccination trials to evaluate their immune response and protective efficacy. However, single in ovo injection of specific DNA vaccine at 18th day of embryonation, regardless using neutral lipid-protected vector or not, failed to trigger significant IgG and IgA immune responses and did not confer protection against C. jejuni colonization in the intestine of chickens. In conclusion, this study demonstrates that the Kozak sequence is critically important for construction of the DNA vaccine expressing prokaryotic gene. The optimal regimen for in ovo vaccination of DNA vaccine for Campylobacter control in poultry needs to be determined in future studies.

Characterization of the emerging multidrug-resistant Salmonella enterica serovar Indiana strains in China.

Emergence of multidrug-resistant (MDR) Salmonella enterica serovar Indiana (S. Indiana), a dominant Salmonella serovar in China, has raised global awareness because the MDR S. Indiana also was rapidly emerged in other countries recently. To improve our understanding of underlying MDR mechanism and evolution of this emerging zoonotic pathogen, here we examined the standard ATCC51959 strain together with 19 diverse and representative Chinese S. Indiana strains by performing comprehensive microbiological, molecular, and comparative genomics analyses. The findings from S1-PFGE, plasmid origin analysis and Southern blotting suggested the MDR phenotype in the majority of isolates was associated with large integron-carrying plasmids. Interestingly, further in-depth analyses of two recently isolated, plasmid-free MDR S. Indiana revealed a long chromosomal class I integron (7.8 kb) that is not linked to the Salmonella Genome Island 1 (SGI1), which is rare. This unique chromosomal integron shares extremely high similarity to that identified in a MDR E. coli plasmid pLM6771 with respect to both genomic organization and sequence identity. Taken together, both plasmid and chromosomal integron I exist in the examined MDR S. Indiana strains. This timely study represents a significant step toward the understanding of molecular basis of the emerging MDR S. Indiana.

Enterobactin-Specific Antibodies Induced by a Novel Enterobactin Conjugate Vaccine.

Enterobactin (Ent)-mediated high-affinity iron acquisition is critical for Gram-negative bacteria to survive in the host. Given the bacteriostatic effect of lipocalin resulting from its potent Ent-binding ability, immune intervention directly targeting Ent is promising for iron-dependent pathogen control. Recently, an Ent conjugate vaccine was reported, but it still has several significant weaknesses. In this study, we sought to develop an innovative Ent conjugate vaccine that can induce a high level of antibodies directed against Ent and to provide solid evidence demonstrating siderophore-binding capacity of Ent-specific antibodies. Using a simple method, we successfully conjugated purified Ent to different carriers, including keyhole limpet hemocyanin (KLH), bovine serum albumin, and CmeC, a vaccine candidate for Campylobacter control. Subcutaneous immunization of rabbits with the KLH-Ent conjugate triggered a strong systemic IgG immune response with an up to 16,384-fold increase in IgG titer directed against whole conjugate and an up to 4,096-fold increase in the level of specific anti-Ent IgG. To evaluate the ability of Ent-specific IgG to bind to the Ent derivatives present in vivo, various Ent derivatives were chemically synthesized and a unique enzyme-linked immunosorbent assay method was developed. The Ent-specific IgG also displayed exceptional reactivity to ferric Ent, a linear trimer of Ent, and different salmochelins. Growth assays further demonstrated that the Ent-specific antibodies significantly inhibited Ent-dependent growth of Campylobacter spp. and Escherichia coli Collectively, this study reports an efficient method to prepare a new type of Ent conjugate vaccines for inducing a high level of Ent-specific antibodies, which can bind to various Ent derivatives and display lipocalin-like bacteriostatic features.IMPORTANCE Ent-mediated high-affinity iron acquisition is a universal and critical contributor for Gram-negative pathogens to survive and infect hosts. Published information has supported an innovative immune intervention strategy that directly targets Ent to starve pathogens by limiting the availability of iron to be utilized. Compared to a recently published Ent conjugate, there are three advantages of the vaccine described in this study: ease of preparation, induction of high titer of anti-Ent IgG, and the ability of Ent-specific antibodies to bind various Ent derivatives, including the salmochelins that help enteric pathogens evade sequestration of siderophores by host lipocalins. In addition, the Ent-specific antibodies were demonstrated to function similarly to lipocalin to interfere with the Ent-dependent growth of Campylobacter and E. coli under iron-restricted conditions. This study has significant potential for broader applications to prevent and control various Gram-negative infections in humans and animals.