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.

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.

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.

Development and Evaluation of Two Live Salmonella-Vectored Vaccines for Campylobacter Control in Broiler Chickens.

Campylobacter is the leading bacterial cause of human enteritis in developed countries. Human campylobacteriosis is commonly associated with the consumption of undercooked, contaminated chicken, a natural host of Campylobacter. Thus, the control of Campylobacter colonization in poultry at the farm level would reduce the risk of human exposure to this pathogen. Vaccination is an attractive intervention measure to mitigate Campylobacter in poultry. Our recent studies have demonstrated that the outer-membrane proteins CmeC (an essential component of CmeABC multidrug efflux pump) and CfrA (ferric enterobactin receptor) are feasible candidates for immune intervention against Campylobacter. By targeting these two promising vaccine candidates, live attenuated Salmonella-vectored vaccines were developed and evaluated in this study. Briefly, the cfrA and cmeC genes were cloned into expression vector pYA3493 and transferred into Salmonella enterica serovar Typhimurium χ8914, the USDA licensed live attenuated vaccine strain. The oral live Salmonella vaccines producing CfrA or CmeC (truncated or full length) were successfully constructed by using delicate molecular manipulation despite the challenge due to the potential toxic effect of the cloned gene product in the Escherichia coli host. Expression and membrane localization of the target protein in the vaccines were confirmed by immunoblotting. The efficacies of the two live vaccines that produce full-length CfrA or CmeC were evaluated by using broiler chickens. However, oral vaccination of chickens failed to trigger significant systemic and intestinal mucosal immune responses and, consequently, did not confer protection against Campylobacter jejuni colonization chickens. The vaccination regimens of the constructed live Salmonella-vectored vaccine need to be optimized in future studies.

A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency in Campylobacter jejuni.

Conjugation is an important mechanism for horizontal gene transfer in Campylobacter jejuni, the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency in Campylobacter spp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC) C. jejuni strain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFC C. jejuni strain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFC C. jejuni strain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme (aka CjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (>5,000-fold). Chromosomal complementation of three diverse HFC C. jejuni strains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest the Escherichia coli-derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency in C. jejuni Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuniIMPORTANCE Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. Campylobacter jejuni, the leading foodborne bacterial organism, displays significant strain diversity due to horizontal gene transfer; however, the molecular components influencing conjugation efficiency in C. jejuni are still largely unknown. In this study, we developed a cotransformation strategy for comparative genomics analysis and successfully identified a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni The new cotransformation strategy developed in this study is also expected to be broadly applied in other naturally competent bacteria for functional comparative genomics research.

Heat Shock-Enhanced Conjugation Efficiency in Standard Campylobacter jejuni Strains.

Campylobacter jejuni, the leading bacterial cause of human gastroenteritis in the United States, displays significant strain diversity due to horizontal gene transfer. Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. It has been observed that heat shock could increase transformation efficiency in some bacteria. In this study, the effect of heat shock on C. jejuni conjugation efficiency and the underlying mechanisms were examined. With a modified Escherichia coli donor strain, different C. jejuni recipient strains displayed significant variation in conjugation efficiency ranging from 6.2 × 10(-8) to 6.0 × 10(-3) CFU per recipient cell. Despite reduced viability, heat shock of standard C. jejuni NCTC 11168 and 81-176 strains (e.g., 48 to 54°C for 30 to 60 min) could dramatically enhance C. jejuni conjugation efficiency up to 1,000-fold. The phenotype of the heat shock-enhanced conjugation in C. jejuni recipient cells could be sustained for at least 9 h. Filtered supernatant from the heat shock-treated C. jejuni cells could not enhance conjugation efficiency, which suggests that the enhanced conjugation efficiency is independent of secreted substances. Mutagenesis analysis indicated that the clustered regularly interspaced short palindromic repeats system and the selected restriction-modification systems (Cj0030/Cj0031, Cj0139/Cj0140, Cj0690c, and HsdR) were dispensable for heat shock-enhanced conjugation in C. jejuni. Taking all results together, this study demonstrated a heat shock-enhanced conjugation efficiency in standard C. jejuni strains, leading to an optimized conjugation protocol for molecular manipulation of this organism. The findings from this study also represent a significant step toward elucidation of the molecular mechanism of conjugative gene transfer in C. jejuni.

Identification and characterization of a periplasmic trilactone esterase, Cee, revealed unique features of ferric enterobactin acquisition in Campylobacter.

Ferric enterobactin (FeEnt) acquisition is a highly efficient and conserved iron scavenging system in Gram-negative bacteria. Recently, we have characterized two FeEnt receptors (CfrA and CfrB) in Campylobacter jejuni and C. coli, the enteric human pathogens that do not produce any siderophores. In this study, whole-genome sequencing and comparative genomic analysis identified a unique Ent trilactone esterase Cee (Cj1376) in C. jejuni. Genomic analysis and biochemical assay strongly suggested that Cee is the sole trilactone esterase in C. jejuni. Thin-layer chromatography and HPLC analyses showed high efficiency of the purified Cee to hydrolyse Ent. Three Cee homologues previously characterized from other bacteria (IroE, IroD and Fes) were also purified and analysed together with Cee, indicating that Cee, Fes and IroD displayed similar hydrolysis dynamics for both apo and ferric forms of Ent while IroE catalysed Ent inefficiently. Unlike cytoplasmic Fes and IroD, Cee is localized in the periplasm as demonstrated by immunoblotting using Cee-specific antibodies. Genetic manipulation of diverse Campylobacter strains demonstrated that Cee is not only essential for CfrB-dependent FeEnt acquisition but also involved in CfrA-dependent pathway. Together, this study identified and characterized a novel periplasmic trilactone esterase and suggested a new model of FeEnt acquisition in Campylobacter.

A single nucleotide in the promoter region modulates the expression of the ß-lactamase OXA-61 in Campylobacter jejuni.

OBJECTIVES: Despite prevalent ß-lactam resistance in Campylobacter jejuni, an important zoonotic enteric pathogen, the molecular basis of ß-lactamase-mediated ß-lactam resistance is still largely unknown. In particular, some C. jejuni strains that carry ß-lactamase gene blaOXA-61 (Cj0299) are still susceptible to ß-lactams with undetected ß-lactamase activity, suggesting blaOXA-61 is subjected to regulation. The objective of this study was to determine the regulatory mechanism of ß-lactamase in C. jejuni. METHODS: An ampicillin-resistant derivative of C. jejuni NCTC 11168 was subjected to whole genome sequencing and comparative genomics analysis. Complementary molecular experiments were further performed to examine the identified regulatory mechanism of blaOXA-61, which included complementation, promoter fusion assay, real-time RT-PCR, natural transformation using a defined PCR fragment, survey of clinical isolates and transcription start site mapping. RESULTS: A single nucleotide mutation (G → T transversion) upstream of blaOXA-61 was identified in the ampicillin-resistant derivative of NCTC 11168. The role of the G → T point mutation in acquired ß-lactam resistance through up-regulating the expression of blaOXA-61 was confirmed by multiple molecular approaches. The G → T transversion in the blaOXA-61 promoter was linked to high-level ß-lactam resistance in C. jejuni isolates. Transcription start site mapping indicated that the G → T transversion restored the TATA box in the -10 region of blaOXA-61. CONCLUSIONS: We demonstrated a novel genetic mechanism of ß-lactamase regulation in C. jejuni in this study, which will provide insights into the regulation and evolution of ß-lactam resistance in Campylobacter.

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.

Identification and characterization of a bile salt hydrolase from Lactobacillus salivarius for development of novel alternatives to antibiotic growth promoters.

Antibiotic growth promoters (AGPs) have been used as feed additives to improve average body weight gain and feed efficiency in food animals for more than 5 decades. However, there is a worldwide trend to limit AGP use to protect food safety and public health, which raises an urgent need to discover effective alternatives to AGPs. The growth-promoting effect of AGPs has been shown to be highly correlated with the decreased activity of intestinal bile salt hydrolase (BSH), an enzyme that is produced by various gut microflora and involved in host lipid metabolism. Thus, BSH inhibitors are likely promising feed additives to AGPs to improve animal growth performance. In this study, the genome of Lactobacillus salivarius NRRL B-30514, a BSH-producing strain isolated from chicken, was sequenced by a 454 GS FLX sequencer. A BSH gene identified by genome analysis was cloned and expressed in an Escherichia coli expression system for enzymatic analyses. The BSH displayed efficient hydrolysis activity for both glycoconjugated and tauroconjugated bile salts, with slightly higher catalytic efficiencies (k(cat)/K(m)) on glycoconjugated bile salts. The optimal pH and temperature for the BSH activity were 5.5 and 41°C, respectively. Examination of a panel of dietary compounds using the purified BSH identified some potent BSH inhibitors, in which copper and zinc have been recently demonstrated to promote feed digestion and body weight gain in different food animals. In sum, this study identified and characterized a BSH with broad substrate specificity from a chicken L. salivarius strain and established a solid platform for us to discover novel BSH inhibitors, the promising feed additives to replace AGPs for enhancing the productivity and sustainability of food animals.

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.

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.

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.

Prevalence, development, and molecular mechanisms of bacteriocin resistance in Campylobacter.

Bacteriocins (BCNs) are antimicrobial peptides produced by bacteria with narrow or broad spectra of antimicrobial activity. Recently, several unique anti-Campylobacter BCNs have been identified from commensal bacteria isolated from chicken intestines. These BCNs dramatically reduced C. jejuni colonization in poultry and are being directed toward on-farm control of Campylobacter. However, no information concerning prevalence, development, and mechanisms of BCN resistance in Campylobacter exists. In this study, susceptibilities of 137 C. jejuni isolates and 20 C. coli isolates to the anti-Campylobacter BCNs OR-7 and E-760 were examined. Only one C. coli strain displayed resistance to the BCNs (MIC, 64 µg/ml), while others were susceptible, with MICs ranging from 0.25 to 4 µg/ml. The C. coli mutants resistant to BCN OR-7 also were obtained by in vitro selection, but all displayed only low-level resistance to OR-7 (MIC, 8 to 16 µg/ml). The acquired BCN resistance in C. coli could be transferred at intra- and interspecies levels among Campylobacter strains by biphasic natural transformation. Genomic examination of the OR-7-resistant mutants by using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to both intrinsic resistance and acquired resistance to the BCNs. Altogether, this study represents the first report of and a major step forward in understanding BCN resistance in Campylobacter, which will facilitate the development of effective BCN-based strategies to reduce the Campylobacter loads in poultry.

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.

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.

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.