Responses of Three Pedicularis Species to Geological and Climatic Changes in the Qinling Mountains and Adjacent Areas in East Asia.

The Qinling Mountains in East Asia serve as the geographical boundary between the north and south of China and are also indicative of climatic differences, resulting in rich ecological and species diversity. However, few studies have focused on the responses of plants to geological and climatic changes in the Qinling Mountains and adjacent regions. Therefore, we investigated the evolutionary origins and phylogenetic relationships of three Pedicularis species in there to provide molecular evidence for the origin and evolution of plant species. Ecological niche modeling was used to predict the geographic distributions of three Pedicularis species during the last interglacial period, the last glacial maximum period, and current and future periods, respectively. Furthermore, the distribution patterns of climate fluctuations and the niche dynamics framework were used to assess the equivalence or difference of niches among three Pedicularis species. The results revealed that the divergence of three Pedicularis species took place in the Miocene and Holocene periods, which was significantly associated with the large-scale uplifts of the Qinling Mountains and adjacent regions. In addition, the geographic distributions of three Pedicularis species have undergone a northward migration from the past to the future. The most important environmental variables affecting the geographic distributions of species were the mean diurnal range and annual mean temperature range. The niche divergence analysis suggested that the three Pedicularis species have similar ecological niches. Among them, P. giraldiana showed the highest niche breadth, covering nearly all of the climatic niche spaces of P. dissecta and P. bicolor. In summary, this study provides novel insights into the divergence and origins of three Pedicularis species and their responses to climate and geological changes in the Qinling Mountains and adjacent regions. The findings have also provided new perspectives for the conservation and management of Pedicularis species.

A Longitudinal Study on Campylobacter in Conventionally Reared Commercial Broiler Flocks in the United States: Prevalence and Genetic Diversity.

Poultry meat contaminated with Campylobacter, a major bacterial cause of foodborne gastroenteritis worldwide, is considered the primary source of human campylobacteriosis. Thus, reduction or elimination of Campylobacter in poultry production will have a significant impact on food safety and public health. Despite the significant progress made over the last decades, many puzzles remain about the epidemiology of Campylobacter on poultry farms, hampering the development of an effective control strategy. This longitudinal study was conducted to determine the prevalence and genetic diversity of Campylobacter in a U.S. commercial broiler production farm system. Cecal contents (15 samples/flock) and boot swabs (3 samples/flock) were collected from approximately 6-wk-old birds from 406 conventional broiler flocks reared in 53 houses on 15 farms (located within a relatively close geographic proximity and managed by the same poultry integrator) for up to eight consecutive production cycles and cultured for Campylobacter. Pulsed-field gel electrophoresis was used to investigate the genetic diversity of the Campylobacter jejuni isolates recovered from the cecal contents. The prevalence of Campylobacter at the farm, house, and flock levels were found to be 93% (14/15), 79% (42/53), and 47% (192/406), respectively. Campylobacter prevalence varied remarkably among different farms and flocks, with some farms or houses testing consistently negative while others being positive all the time over the entire study period. Campylobacter isolation rate changed significantly by sample type (higher by cecal contents vs. boot swabs) and season/production cycle (higher in spring vs. other seasons). The majority (88%; 2364/2675) of the isolates were identified as C. jejuni, and almost all the rest (11%; 303/2675) were Campylobacter coli. Genotyping showed limited diversity within a flock and suggested persistence of some C. jejuni clones over multiple production cycles on the same farm. In conclusion, this study indicated that although Campylobacter prevalence was overall high, there were marked differences in the prevalence among the broiler flocks or farms tested. Future studies aimed at identification of potential risk factors associated with differential Campylobacter status are warranted in order to develop effective on-farm interventions.

Estudio longitudinal sobre Campylobacter en parvadas comerciales de pollo de engorde criados convencionalmente en los Estados Unidos: prevalencia y diversidad genética. Los productos cárnicos de origen avícola contaminado con Campylobacter, que es una importante causa bacteriana de gastroenteritis transmitida por alimentos en todo el mundo, se consideran la principal fuente de campilobacteriosis humana. Por lo tanto, la reducción o eliminación de Campylobacter en la producción avícola tendrá un impacto significativo en la seguridad alimentaria y en la salud pública. A pesar de los importantes avances realizados en las últimas décadas, persisten muchos enigmas sobre la epidemiología de Campylobacter en las granjas avícolas, lo que obstaculiza el desarrollo de una estrategia de control eficaz. Este estudio longitudinal se realizó para determinar la prevalencia y la diversidad genética de Campylobacter en un sistema de granja de producción comercial de pollos de engorde en los Estados Unidos. Se recogieron contenidos cecales (15 muestras/parvada) y cubre botas de arrastre (tres muestras/parvada) de aves de aproximadamente seis semanas de edad de 406 parvadas de pollos de engorde convencionales criadas en 53 casetas de 15 granjas (ubicadas dentro de una proximidad geográfica relativamente cercana y manejadas por el mismo integrador avícola) durante ocho ciclos de producción consecutivos y con cultivo para Campylobacter. Se utilizó electroforesis en gel de campo con pulsasiones para investigar la diversidad genética de los aislados de Campylobacter jejuni recuperados del contenido cecal. Se encontró que la prevalencia de Campylobacter a nivel de granja, caseta y parvada era del 93% (14/15), 79% (42/53) y 47% (192/406), respectivamente. La prevalencia de Campylobacter varió notablemente entre diferentes granjas y rebaños, y algunas granjas o casetas dieron resultados consistentemente negativos mientras que otras dieron positivo todo el tiempo durante todo el período del estudio. La tasa de aislamiento de Campylobacter cambió significativamente según el tipo de muestra (mayor con muestras de contenido cecal en comparación con los cubre botas de arrastre) y la estación/ciclo de producción (mayor en primavera frente a otras estaciones). La mayoría (88%; 2364/2675) de los aislados se identificaron como C. jejuni, y casi todo el resto (11%; 303/2675) fueron Campylobacter coli. La genotipificación mostró una diversidad limitada dentro de una parvada y sugirió la persistencia de algunos clones de C. jejuni durante múltiples ciclos de producción en la misma granja. En conclusión, este estudio indicó que, aunque la prevalencia de Campylobacter fue alta en general, hubo marcadas diferencias en la prevalencia entre las parvadas o granjas de pollos de engorde analizadas. Se justifica la conducción de estudios futuros destinados a identificar posibles factores de riesgo asociados con el estado diferencial de Campylobacter para desarrollar intervenciones efectivas en las granjas.

Structural basis of DNA recognition of the Campylobacter jejuni CosR regulator.

Campylobacter jejuni is a foodborne pathogen commonly found in the intestinal tracts of animals. This pathogen is a leading cause of gastroenteritis in humans. Besides its highly infectious nature, C. jejuni is increasingly resistant to a number of clinically administrated antibiotics. As a consequence, the Centers for Disease Control and Prevention has designated antibiotic-resistant Campylobacter as a serious antibiotic resistance threat in the United States. The C. jejuni CosR regulator is essential to the viability of this bacterium and is responsible for regulating the expression of a number of oxidative stress defense enzymes. Importantly, it also modulates the expression of the CmeABC multidrug efflux system, the most predominant and clinically important system in C. jejuni that mediates resistance to multiple antimicrobials. Here, we report structures of apo-CosR and CosR bound with a 21 bp DNA sequence located at the cmeABC promotor region using both single-particle cryo-electron microscopy and X-ray crystallography. These structures allow us to propose a novel mechanism for CosR regulation that involves a long-distance conformational coupling and rearrangement of the secondary structural elements of the regulator to bind target DNA. IMPORTANCE: Campylobacter jejuni has emerged as an antibiotic-resistant threat worldwide. CosR is an essential regulator for this bacterium and is important for Campylobacter adaptation to various stresses. Here, we describe the structural basis of CosR binding to target DNA as determined by cryo-electron microscopy and X-ray crystallography. Since CosR is a potential target for intervention, our studies may facilitate the development of novel therapeutics to combat C. jejuni infection.

Nonparametric bootstrap methods for interval estimation of the area under the ROC curve with correlated diagnostic test data: application to whole-virus ELISA testing in swine.

Developing and evaluating novel diagnostic assays are crucial components of contemporary diagnostic research. The receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) are frequently used to evaluate diagnostic assays' performance. The variation in AUC estimation can be quantified nonparametrically using resampling methods, such as bootstrapping, and then used to construct interval estimation for the AUC. When multiple observations are observed from the same subject, which is very common in veterinary diagnostic tests evaluation experiments, a traditional bootstrap-based method can fail to provide valid interval estimations of AUC. In particular, the traditional method does not account for the correlation among data observations and could result in interval estimation that fails to cover the true AUC adequately at the desired confidence level. In this paper, we proposed two novel methods to calculate the confidence interval of the AUC for correlated diagnostic test data based on cluster bootstrapping and hierarchical bootstrapping, respectively. Our simulation studies showed that both proposed methods had adequate coverage probabilities which were higher than the existing traditional method when there were intra-subject correlations. We also discussed applying the proposed methods to evaluate a novel whole-virus ELISA (wv-ELISA) diagnostic assay in detecting porcine parainfluenza virus type-1 antibodies in swine serum.

Fecal Microbiota Transplantation Reduces Campylobacter jejuni Colonization in Young Broiler Chickens Challenged by Oral Gavage but Not by Seeder Birds.

Campylobacter spp., particularly C. jejuni and C. coli, are major food safety concerns, transmitted to humans mainly via contaminated poultry meat. In a previous study, we found that some commercial broiler farms consistently produced Campylobacter-free flocks while others consistently reared Campylobacter-colonized flocks, and significant differences in the gut microbiota compositions between the two types of farm categories were revealed. Therefore, we hypothesized that gut microbiota influences Campylobacter colonization in poultry and that the microbiota from Campylobacter-free flocks may confer colonization resistance to Campylobacter in the chicken intestine. In this study, two fecal microbiota transplantation (FMT) trials were performed to test the hypothesis. Newly hatched chicks were given FMT via oral gavage of the cecal content of Campylobacter-free adult chickens (treatment groups) or PBS (control groups) before the feed consumption. Approximately two weeks after the FMT, the birds were challenged with C. jejuni either by oral gavage (trial 1) or by co-mingling with Campylobacter-colonized seeder birds (trial 2) to evaluate the potential protective effect of the FMT. Cecal contents were collected (3 times, 5 days apart) to determine the Campylobacter colonization levels via culture and microbiota compositions via 16S rRNA gene sequencing. FMT reduced cecal Campylobacter colonization significantly (log10 1.2-2.54 CFU/g) in trial 1 but not in trial 2, although FMT significantly impacted the diversity and compositions of the gut microbiota in both trials. Several genera, such as Butyricimonas, Parabacteroides, Parasutterella, Bilophila, Fournierella, Phascolarctobacterium, and Helicobacter, had increased abundance in the FMT-treated groups in both trials. Furthermore, Campylobacter abundance was found to be negatively correlated with the Escherichia and Ruminococcus_torques_group genera. These findings indicate that even though FMT with adult cecal microbiota can positively affect the subsequent development of the gut microbiota in young broilers, its inhibitory effect on Campylobacter colonization varies and appears to be influenced by the challenge models.

[A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing].

During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.

Direct interaction of small non-coding RNAs CjNC140 and CjNC110 optimizes expression of key pathogenic phenotypes of Campylobacter jejuni.

Small non-coding RNAs (sRNAs) are important players in modulating gene expression in bacterial pathogens, but their functions are largely undetermined in Campylobacter jejuni, an important cause of foodborne gastroenteritis in humans. In this study, we elucidated the functions of sRNA CjNC140 and its interaction with CjNC110, a previously characterized sRNA involved in the regulation of several virulence phenotypes of C. jejuni. Inactivation of CjNC140 increased motility, autoagglutination, L-methionine concentration, autoinducer-2 production, hydrogen peroxide resistance, and early chicken colonization, indicating a primarily inhibitory role of CjNC140 for these phenotypes. Apart from motility, all these effects directly contrasted the previously demonstrated positive regulation by CjNC110, suggesting that CjNC110 and CjNC140 operate in an opposite manner to modulate physiologic processes in C. jejuni. RNAseq and northern blotting further demonstrated that expression of CjNC140 increased in the absence of CjNC110, while expression of CjNC110 decreased in the absence of CjNC140, suggesting a possibility of their direct interaction. Indeed, electrophoretic mobility shift assay demonstrated a direct binding between the two sRNAs via GA- (CjNC110) and CU- (CjNC140) rich stem-loops. Additionally, RNAseq and follow-up experiments identified that CjNC140 positively regulates p19, which encodes a key iron uptake transporter in Campylobacter. Furthermore, computational analysis revealed both CjNC140 and CjNC110 are highly conserved in C. jejuni, and the predicted secondary structures support CjNC140 as a functional homolog of the iron regulatory sRNA, RyhB. These findings establish CjNC140 and CjNC110 as a key checks-and- balances mechanism in maintaining homeostasis of gene expression and optimizing phenotypes critical for C. jejuni pathobiology. IMPORTANCE Gene regulation is critical to all aspects of pathogenesis of bacterial disease, and small non-coding RNAs (sRNAs) represent a new frontier in gene regulation of bacteria. In Campylobacter jejuni, the role of sRNAs remains largely unexplored. Here, we investigate the role of two highly conserved sRNAs, CjNC110 and CjNC140, and demonstrate that CjNC140 displays a primarily inhibitory role in contrast to a primarily activating role for CjNC110 for several key virulence-associated phenotypes. Our results also revealed that the sRNA regulatory pathway is intertwined with the iron uptake system, another virulence mechanism critical for in vivo colonization. These findings open a new direction for understanding C. jejuni pathobiology and identify potential targets for intervention for this major foodborne pathogen.

Deciphering the Association between Campylobacter Colonization and Microbiota Composition in the Intestine of Commercial Broilers.

Campylobacter is a major food safety concern and is transmitted mainly via poultry meat. We previously found that some commercial broiler farms consistently produced Campylobacter-negative flocks while others were consistently Campylobacter-positive for consecutive production cycles although the farms operated under similar management practices. We hypothesized that this difference in Campylobacter colonization might be associated with the gut microbiota composition. To address this, six commercial broiler farms were selected based on their Campylobacter status (three negative and three positive) to evaluate the microbiota differences between each farm category. For each farm on each production cycle (2-3 cycles), 40 ceca collected from five-week-old broilers were processed for microbiota analysis via 16S rRNA gene sequencing. Cecal microbiota species richness, phylogenetic diversity, community structure, and composition of Campylobacter-positive farms were noticeably different from those of Campylobacter-negative farms. Rikenella, Methanocorpusculum, Barnesiella, Parasutterella, and Helicobacter were significantly more abundant among Campylobacter-positive farms. In contrast, Ruminococcaceae, Streptococcus, Escherichia, Eggerthellaceae, Lactobacillus, Monoglobus, and Blausia were more abundant in Campylobacter-negative farms. Eggerthellaceae, Clostridia, Lachnospiraceae, Lactobacillus, Monoglobus, and Parabacteroides were significantly negatively correlated with Campylobacter abundance. These findings suggest that specific members of cecal microbiota may influence Campylobacter colonization in commercial broilers and may be further explored to control Campylobacter in poultry.

Cryo-Electron Microscopy Structures of a Campylobacter Multidrug Efflux Pump Reveal a Novel Mechanism of Drug Recognition and Resistance.

Campylobacter jejuni is a bacterium that is commonly present in the intestinal tracts of animals. It is also a major foodborne pathogen that causes gastroenteritis in humans. The most predominant and clinically important multidrug efflux system in C. jejuni is the CmeABC (Campylobacter multidrug efflux) pump, a tripartite system that includes an inner membrane transporter (CmeB), a periplasmic fusion protein (CmeA), and an outer membrane channel protein (CmeC). This efflux protein machinery mediates resistance to a number of structurally diverse antimicrobial agents. A recently identified CmeB variant, termed resistance enhancing CmeB (RE-CmeB), can increase its multidrug efflux pump activity, likely by influencing antimicrobial recognition and extrusion. Here, we report structures of RE-CmeB in its apo form as well as in the presence of four different drugs by using single-particle cryo-electron microscopy (cryo-EM). Coupled with mutagenesis and functional studies, this structural information allows us to identify critical amino acids that are important for drug resistance. We also report that RE-CmeB utilizes a somewhat unique subset of residues to bind different drugs, thereby optimizing its ability to accommodate different compounds with distinct scaffolds. These findings provide insights into the structure-function relationship of this newly emerged antibiotic efflux transporter variant in Campylobacter. IMPORTANCE Campylobacter jejuni has emerged as one of the most problematic and highly antibiotic-resistant pathogens, worldwide. The Centers for Disease Control and Prevention have designated antibiotic-resistant C. jejuni as a serious antibiotic resistance threat in the United States. We recently identified a C. jejuni resistance enhancing CmeB (RE-CmeB) variant that can increase its multidrug efflux pump activity and confers an exceedingly high-level of resistance to fluoroquinolones. Here, we report the cryo-EM structures of this prevalent and clinically important C. jejuni RE-CmeB multidrug efflux pump in both the absence and presence of four antibiotics. These structures allow us to understand the action mechanism for multidrug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

Growth kinetics and fitness of fluoroquinolone resistant and susceptible Campylobacter jejuni strains of cattle origin.

Human enterocolitis is frequently caused by the Gram-negative microaerobic bacterium Campylobacter jejuni. Macrolides (e.g., erythromycin) and fluoroquinolones (FQs) (e.g., ciprofloxacin) are the preferred antibiotics for the treatment of human campylobacteriosis. Rapid emergence of FQ-resistant (FQ-R) Campylobacter during treatment with FQ antimicrobials is well known to occur in poultry. Cattle is also an important reservoir of Campylobacter for humans, and FQ-R Campylobacter from cattle has become highly prevalent in recent years. Even though the selection pressure may have contributed to the expansion of FQ-R Campylobacter, the actual impact of this factor appears to be rather low. In this study, we examined the hypothesis that the fitness of FQ-R Campylobacter may have also played a role in the rise seen in FQ-R Campylobacter isolates by employing a series of in vitro experiments in MH broth and bovine fecal extract. First, it was shown that FQ-R and FQ-susceptible (FQ-S) C. jejuni strains of cattle origin had comparable growth rates when individually cultured in both MH broth and the fecal extract with no antibiotic present. Interestingly, FQ-R strains had small but statistically significant increases over FQ-S strains in growth in competition experiments performed in mixed cultures with no antibiotic present. Lastly, it was observed that FQ-S C. jejuni strains developed resistance to ciprofloxacin more readily at high initial bacterial cell density (107 CFU/mL) and when exposed to low levels of the antibiotic (2-4 µg/mL) compared with that at a low level of initial bacterial cell density (105 CFU/mL) and exposure to a high level of ciprofloxacin (20 µg/mL) in both MH broth and the fecal extract. Altogether, these findings indicate that even though FQ-R C. jejuni of cattle origin may have a slightly higher fitness advantage over the FQ-S population, the emergence of FQ-R mutants from susceptible strains is primarily dictated by the bacterial cell density and the antibiotic concentration exposed under in vitro condition. These observation may also provide plausible explanations for the high prevalence of FQ-R C. jejuni in cattle production due to its overall fit nature in the absence of antibiotic selection pressure and for the paucity of development of FQ-R C. jejuni in the cattle intestine in response to FQ-treatment, as observed in our recent studies.

Recent advances in the chemistry of aryltriazene.

Triazene is one of the most versatile building blocks in organic synthesis. Generally, it is viewed as a safe equivalent of diazonium salt, thus immediately finding numerous applications in preparative chemistry and medicinal chemistry. Besides, it can be used as a removable directing group in C-H functionalization or play a smart role as a precursor for aryl cation/radical generation. In this review, we will highlight recent noteworthy developments in this field.

Influence of Single Dose Enrofloxacin Injection on Development of Fluoroquinolone Resistance in Campylobacter jejuni in Calves.

Fluoroquinolone (FQ) resistance in a major foodborne bacterial pathogen, Campylobacter jejuni, derived from cattle has recently become prevalent and poses a significant public health concern. However, the underlying factors for this increase are not entirely clear. To evaluate the effect of enrofloxacin treatment on FQ-resistance development in C. jejuni, 35 commercial calves were equally divided into five groups (Groups 1-5) and were orally inoculated with FQ-susceptible (FQ-S) C. jejuni. Eight days later, Groups 4 and 5 were challenged with Mannheimia haemolytica via a transtracheal route to induce a respiratory disease; after 8 days, Groups 2, 3, 4, and 5 were injected subcutaneously with enrofloxacin (7.5 mg/kg for Groups 2 and 4, and 12.5 mg/kg for Groups 3 and 5). Colonization levels by FQ-resistant (FQ-R) and FQ-S Campylobacter in rectal feces were determined via differential culture throughout the experiment. Before oral inoculation with C. jejuni, only five calves were naturally colonized by Campylobacter, four of which were also colonized by FQ-R C. jejuni (three in Group 1 and one in Group 3). Soon after the oral inoculation, almost all calves in the groups became stably colonized by FQ-S C. jejuni (~3-6 log10 CFU/g), except that the four calves that were pre-colonized before inoculation remained positive with both FQ-R and FQ-S C. jejuni. Following enrofloxacin administration, C. jejuni colonization declined sharply and rapidly in all treated groups to undetectable levels; however, the vast majority of the animals were recolonized by C. jejuni at comparable levels 72 h after the treatment. Notably, no FQ-R C. jejuni was detected in any of the calves that received enrofloxacin, regardless of the drug dose used or disease status of the animals. The lack of detection of FQ-R C. jejuni was likely due to the localized high concentration of the antibiotic in the intestine, which may have prevented the emergence of the FQ-R mutant. These findings indicate that single-dose enrofloxacin use in cattle poses a low risk for selection of de novo FQ-R mutants in C. jejuni.

Redox-Neutral Generation of Iminyl Radicals by N-Heterocyclic Carbene Catalysis: Rapid Access to Phenanthridines from Vinyl Azides.

An N-heterocyclic carbene-catalyzed oxidant-, metal- and light-free iminyl radical generation pathway stemming from the reaction of vinyl azide and α-bromo ester is uncovered. This newly developed methodology is successfully applied to the redox-neutral construction of a number of diversified phenanthridine derivatives with nice functional group compatibility. Insights from the mechanism study reveal that this NHC-catalyzed transformation potentially proceeds through an alkyl radical addition-initiated HAS process, with the iminyl radical as an active intermediate.

Bacillus subtilis PS-216 Spores Supplemented in Broiler Chicken Drinking Water Reduce Campylobacter jejuni Colonization and Increases Weight Gain.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis, or campylobacteriosis, in humans worldwide, and poultry serves as a major source of infection. To reduce the risk associated with C. jejuni transmission via poultry meat, effective interventions during poultry production are needed, and the use of probiotics is a promising approach. In this study, 15 Bacillus subtilis strains were initially screened for their anti-Campylobacter activities. B. subtilis PS-216 strain demonstrated the best anti-Campylobacter activity against 15 C. jejuni isolates when examined using in vitro co-cultures. To evaluate the suitability of B. subtilis PS-216 for probiotic use, its susceptibility to eight clinically important antimicrobials and simulated gastric conditions was investigated. B. subtilis PS-216 was sensitive to all of the tested antibiotics. Although vegetative cells were sensitive to gastric conditions, B. subtilis PS-216 spores were highly resistant. We further evaluated the use of a B. subtilis PS-216 spore preparation (2.5 × 106 CFU/mL water) to prevent and/or reduce C. jejuni colonization in broiler chickens in vivo. Compared to the untreated group, significantly lower Campylobacter counts were detected in caeca of broilers continuously treated with B. subtilis PS-216 spores in their drinking water. Furthermore, broilers continuously treated with B. subtilis PS-216 spores showed improved weight gain, compared to the control group. Together, these results demonstrate the potential of B. subtilis PS-216 for use in poultry to reduce C. jejuni colonization and improve weight gain.

Construction of N-Polyheterocycles by N-Heterocyclic Carbene Catalysis via a Regioselective Intramolecular Radical Addition/Cyclization Cascade.

N-Polyheterocycles are rapidly accessed by N-heterocyclic carbene (NHC) catalysis through regioselective sequential radical addition/cyclization in the absence of any metals or oxidants. The transformation occurs under mild conditions and enjoys a wide substrate scope with excellent functional group compatibility. Furthermore, a gram-scale synthesis is also conducted. Preliminary mechanistic studies reveal the potential involvement of an NHC radical cation intermediate.

Nrf2 Activation Protects Against Organic Dust and Hydrogen Sulfide Exposure Induced Epithelial Barrier Loss and K. pneumoniae Invasion.

Agriculture workers report various respiratory symptoms owing to occupational exposure to organic dust (OD) and various gases. Previously, we demonstrated that pre-exposure to hydrogen sulfide (H2S) alters the host response to OD and induces oxidative stress. Nrf2 is a master-regulator of host antioxidant response and exposures to toxicants is known to reduce Nrf2 activity. The OD exposure-induced lung inflammation is known to increase susceptibility to a secondary microbial infection. We tested the hypothesis that repeated exposure to OD or H2S leads to loss of Nrf2, loss of epithelial cell integrity and that activation of Nrf2 rescues this epithelial barrier dysfunction. Primary normal human bronchial epithelial (NHBE) cells or mouse precision cut-lung slices (PCLS) were treated with media, swine confinement facility organic dust extract (ODE) or H2S or ODE+H2S for one or five days. Cells were also pretreated with vehicle control (DMSO) or RTA-408, a Nrf2 activator. Acute exposure to H2S and ODE+H2S altered the cell morphology, decreased the viability as per the MTT assay, and reduced the Nrf2 expression as well as increased the keap1 levels in NHBE cells. Repeated exposure to ODE or H2S or ODE+H2S induced oxidative stress and cytokine production, decreased tight junction protein occludin and cytoskeletal protein ezrin expression, disrupted epithelial integrity and resulted in increased Klebsiella pneumoniae invasion. RTA-408 (pharmacological activator of Nrf2) activated Nrf2 by decreasing keap1 levels and reduced ODE+H2S-induced changes including reversing loss of barrier integrity, inflammatory cytokine production and microbial invasion in PCLS but not in NHBE cell model. We conclude that Nrf2 activation has a partial protective function against ODE and H2S.

Effect of Danofloxacin Treatment on the Development of Fluoroquinolone Resistance in Campylobacter jejuni in Calves.

Campylobacter is a leading cause of foodborne gastroenteritis. Recent studies have indicated a rise in fluoroquinolone-resistant (FQ-R) Campylobacter in cattle, where FQ is used to control bovine respiratory disease (BRD). To assess the effect of danofloxacin treatment on the development of FQ-resistance in C. jejuni, 30 commercial calves were divided into Group 1, Group 2, and Group 3 (n = 10), and were all inoculated orally with FQ-susceptible (FQ-S) C. jejuni; seven days later, Group 3 was challenged with transtracheal Mannheimia haemolytica, and one week later, Group 2 and Group 3 were injected subcutaneously with danofloxacin. Rectal feces were collected to determine relative percentages of FQ-R Campylobacter via culture. Before oral inoculation with C. jejuni, 87% of calves were naturally colonized by FQ-R C. jejuni. Two days after the inoculation, FQ-R C. jejuni decreased substantially in the majority of calves. Within 24 h of danofloxacin injection, almost all C. jejuni populations shifted to an FQ-R phenotype in both FQ-treated groups, which was only transitory, as FQ-S strains became predominant during later periods. Genotyping indicated that the spike seen in FQ-R C. jejuni populations following the injection was due mainly to enrichment of preexisting FQ-R C. jejuni, rather than development of de novo FQ resistance in susceptible strains. These results provide important insights into the dynamic changes of FQ-resistant Campylobacter in cattle in response to FQ treatment.

Comparisons of plasma and fecal pharmacokinetics of danofloxacin and enrofloxacin in healthy and Mannheimia haemolytica infected calves.

Danofloxacin and enrofloxacin are fluoroquinolones (FQs) used to treat and control bovine respiratory disease (BRD) complex. While low toxicity, high bactericidal activity, and availability in single and multiple dosing regimens make them preferable, the increasing incidence of FQ-resistance in foodborne pathogens and effects on gut microbiota necessitate evaluating their pharmacokinetics (PKs). The objective of this study was to determine the exposure level of gut microbiota to subcutaneously administered FQs and compare their PKs between plasma and feces in healthy and Mannheimia haemolytica infected calves. A single dose of danofloxacin (8 mg/kg), low dose (7.5 mg/kg), or high dose (12.5 mg/kg) of enrofloxacin was administered to calves. Blood and feces were collected from calves under experimental conditions over 48 h, and FQ concentrations were measured using Ultra High-Pressure Liquid Chromatography. While moderate BRD signs were exhibited in most calves in the infected cohorts, the plasma PKs were similar between healthy and sick calves. However, the fecal danofloxacin concentration was lower in the BRD group (area under concentration-time curve [AUCinf], BRD median = 2627, healthy median = 2941 h*µg/mL, adj.P = 0.005). The dose normalized plasma and fecal danofloxacin concentrations were higher than those of enrofloxacin and its metabolite ciprofloxacin. Further, FQs had several fold higher overall concentrations in feces than in plasma in both groups. In conclusion, parenterally administered FQs expose gut microbiota to high concentrations of the antibiotics.

Danofloxacin Treatment Alters the Diversity and Resistome Profile of Gut Microbiota in Calves.

Fluoroquinolones, such as danofloxacin, are used to control bovine respiratory disease complex in beef cattle; however, little is known about their effects on gut microbiota and resistome. The objectives were to evaluate the effect of subcutaneously administered danofloxacin on gut microbiota and resistome, and the composition of Campylobacter in calves. Twenty calves were injected with a single dose of danofloxacin, and ten calves were kept as a control. The effects of danofloxacin on microbiota and the resistome were assessed using 16S rRNA sequencing, quantitative real-time PCR, and metagenomic Hi-C ProxiMeta. Alpha and beta diversities were significantly different (p < 0.05) between pre-and post-treatment samples, and the compositions of several bacterial taxa shifted. The patterns of association between the compositions of Campylobacter and other genera were affected by danofloxacin. Antimicrobial resistance genes (ARGs) conferring resistance to five antibiotics were identified with their respective reservoirs. Following the treatment, some ARGs (e.g., ant9, tet40, tetW) increased in frequencies and host ranges, suggesting initiation of horizontal gene transfer, and new ARGs (aac6, ermF, tetL, tetX) were detected in the post-treatment samples. In conclusion, danofloxacin induced alterations of gut microbiota and selection and enrichment of resistance genes even against antibiotics that are unrelated to danofloxacin.

Enrofloxacin Alters Fecal Microbiota and Resistome Irrespective of Its Dose in Calves.

Enrofloxacin is a fluoroquinolone drug used to prevent and control bovine respiratory disease (BRD) complex in multiple or single doses, ranging from 7.5 to 12.5 mg/kg body weight. Here, we examined the effects of high and low doses of a single subcutaneously injected enrofloxacin on gut microbiota and resistome in calves. Thirty-five calves sourced for this study were divided into five groups: control (n = 7), two low dose groups (n = 14, 7.5 mg/kg), and two high dose groups (n = 14, 12.5 mg/kg). One group in the low and high dose groups was challenged with Mannheimia haemolytica to induce BRD. Both alpha and beta diversities were significantly different between pre- and post-treatment microbial communities (q < 0.05). The high dose caused a shift in a larger number of genera than the low dose. Using metagenomic ProxiMeta Hi-C, 32 unique antimicrobial resistance genes (ARGs) conferring resistance to six antibiotic classes were detected with their reservoirs, and the high dose favored clonal expansion of ARG-carrying bacterial hosts. In conclusion, enrofloxacin treatment can alter fecal microbiota and resistome irrespective of its dose. Hi-C sequencing provides significant benefits for unlocking new insights into the ARG ecology of complex samples; however, limitations in sample size and sequencing depth suggest that further work is required to validate the findings.