Pathogenic and multidrug-resistant Escherichia fergusonii from broiler chicken.

An Escherichia spp. isolate, ECD-227, was previously identified from the broiler chicken as a phylogenetically divergent and multidrug-resistant Escherichia coli possessing numerous virulence genes. In this study, whole genome sequencing and comparative genome analysis was used to further characterize this isolate. The presence of known and putative antibiotic resistance and virulence open reading frames were determined by comparison to pathogenic (E. coli O157:H7 TW14359, APEC O1:K1:H7, and UPEC UTI89) and nonpathogenic species (E. coli K-12 MG1655 and Escherichia fergusonii ATCC 35469). The assembled genome size of 4.87 Mb was sequenced to 18-fold depth of coverage and predicted to contain 4,376 open reading frames. Phylogenetic analysis of 537 open reading frames present across 110 enteric bacterial species identifies ECD-227 to be E. fergusonii. The genome of ECD-227 contains 5 plasmids showing similarity to known E. coli and Salmonella enterica plasmids. The presence of virulence and antibiotic resistance genes were identified and localized to the chromosome and plasmids. The mutation in gyrA (S83L) involved in fluoroquinolone resistance was identified. The Salmonella-like plasmids harbor antibiotic resistance genes on a class I integron (aadA, qacEΔ-sul1, aac3-VI, and sulI) as well as numerous virulence genes (iucABCD, sitABCD, cib, traT). In addition to the genome analysis, the virulence of ECD-227 was evaluated in a 1-d-old chick model. In the virulence assay, ECD-227 was found to induce 18 to 30% mortality in 1-d-old chicks after 24 h and 48 h of infection, respectively. This study documents an avian multidrug-resistant and virulent E. fergusonii. The existence of several resistance genes to multiple classes of antibiotics indicates that infection caused by ECD-227 would be difficult to treat using antimicrobials currently available for poultry.

Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry.

A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential.

Growth performance, meat quality, and gut microflora of broiler chickens fed with cranberry extract.

Cranberry fruit components have been reported to have antimicrobial activities against a variety of pathogenic bacteria and to be beneficial for human health. Studies on their effects are very limited in animals and especially in chickens. This study investigated the effect of feed supplementation with a commercial cranberry fruit extract (CFE) on the performance, breast meat quality, and intestinal integrity of broiler chickens. Twelve hundred male 1-d-old broiler chicks were allocated randomly to CFE treatments at 0, 40, 80, or 160 mg/kg of feed from d 0 to 35. Cloacal and cecal samples were collected weekly to evaluate the influence of treatments on the intestinal population of generic Escherichia coli, Clostridium perfringens, Enterococcus spp., and Lactobacillus spp. At d 35, BW were 1.62, 1.60, 1.61, and 1.64 kg for the control birds and birds fed 40, 80, and 160 mg of CFE/kg of feed, respectively. Feed intake ranged from 2.7 to 2.8 kg and feed efficiency from 1.8 to 1.9 g of feed/g of BW. However, the treatment effects on bird performance were not statistically significant (P > 0.05). The mortality rate tended to be lower (P = 0.09) in birds fed 40 mg of CFE/kg of feed. Feed supplementation with CFE did not significantly alter any broiler meat properties evaluated when compared with the control diet (P > 0.05). At d 28, the populations of Enterococcus spp. in cecal and cloacal samples were significantly lower (P < 0.05) in birds receiving CFE at 160 mg/kg of feed than the other groups. No significant differences were noted between the control and the treatment groups for general health and intestinal integrity (P > 0.05). These findings suggest that more studies are needed to investigate potential beneficial effects of CFE or its derivatives in broiler production.

Veterinary pharmaceuticals and antibiotic resistance of Escherichia coli isolates in poultry litter from commercial farms and controlled feeding trials.

Veterinary pharmaceuticals are commonly used in poultry farming to prevent and treat microbial infections as well as to increase feed efficiency, but their use has created public and environmental health concerns. Poultry litter contains antimicrobial residues and resistant bacteria; when applied as fertilizer, the level and effects of these pharmaceuticals and antimicrobial-resistant bacteria in the environment are of concern. The purpose of this study was to investigate poultry litter for veterinary pharmaceuticals and resistance patterns of Escherichia coli. Litter samples were collected from controlled feeding trials and from commercial farms. Feed additives bacitracin, chlortetracycline, monensin, narasin, nicarbazin, penicillin, salinomycin, and virginiamycin, which were present in the feed on commercial farms and added to the feed in the controlled trials, were extracted in methanol and analyzed by liquid chromatography-mass spectrometry techniques. Sixty-nine E. coli were isolated and identified by API 20E. The susceptibility of the isolates to antibiotics was determined using Avian plates and the Sensititer automated system. This study confirmed the presence of antimicrobial residues in broiler litter from controlled environments as well as commercial farms, ranging from 0.07 to 66 mg/L depending on the compound. Concentrations of individual residues were higher in litter from controlled feeding trials than those from commercial farms. All E. coli isolates from commercial farms were multiresistant to at least 7 antibiotics. Resistance to beta-lactam antibiotics (amoxicillin, ceftiofur), tetracyclines, and sulfonamides was the most prevalent. This study concluded that broiler litter is a source of antimicrobial residues and represents a reservoir of multiple antibiotic-resistant E. coli.

Interleukin-1beta up-regulates expression of neurofilament light in human neuronal cells.

Elevated expression of interleukin-1 (IL-1beta), a pro-inflammatory cytokine secreted by activated microglia, is a pathogenic marker of numerous neurodegenerative processes including Alzheimer's disease (AD). We have characterized a link between IL-1beta and the 68-kDa neurofilament light (NF-L) protein, which is a major component of the neuronal cytoskeleton. Using human brain aggregate cultures, we found that IL-1beta treatment significantly increased NF-L expression in primary neurons. Analysis of mRNA levels demonstrated elevated NF-L expression within 72 h while imaging of neurons by immunofluorescent staining for NF-L confirmed IL-1beta-induced NF-L protein expression. These observations suggest a potential inflammatory-induced mechanism for deregulation of an important cytoskeletal protein, NF-L, possibly leading to neuronal dysfunction.

CPI-1189 attenuates effects of suspected neurotoxins associated with AIDS dementia: a possible role for ERK activation.

Individuals infected with the human immunodeficiency virus (HIV) often experience a dementia characterized by mental slowing and memory loss. Motor dysfunction may also accompany this condition. The pathogenesis of the dementia is not known, but microscopic examination of brain tissue from those afflicted shows evidence of chronic inflammation, reactive gliosis and cell death. Neurotoxic factors produced from activated macrophage or microglial cells such as tumor necrosis factor-alpha (TNFalpha), gp120 and quinolinic acid have been implicated as agents for the cell death which often appears to occur by an apoptotic mechanism. CPI-1189, a drug currently undergoing clinical evaluation as a treatment for the dementia associated with AIDS, is shown in this paper to mitigate apoptosis induced by TNFalpha, gp120, and necrosis induced by quinolinic acid. In addition, CPI-1189 mitigates the cell death produced by supernatants from cultured macrophages obtained from patients with AIDS dementia. The exact mechanism by which CPI-1189 prevents neurotoxicity is not known; however, protection from TNFalpha and supernatant-induced toxicity does not appear to involve NFkappaB translocation, and appears to be associated with an increase in activated ERK-MAP kinase. These findings may have implications for other neurological diseases where apoptotic cell death contributes to neurodegeneration.

Elevation of CD69+ monocyte/macrophages in patients with Alzheimer's disease.

In this report, we examined the presence of the activation marker, CD69, on monocytes derived from patients with Alzheimer's disease (AD). We have previously shown that patients with AIDS dementia had an elevated percentage of a CD14+/CD69+ subset and that conditioned media from these M/M phi cultures were toxic to neural cultures. We therefore postulated that patients with AD might likewise have a higher monocyte subset and that this would be associated with neural toxicity. Flow analysis showed that AD patients (n = 13) had a higher percentage of CD69+ M/M phi over age matched controls (n = 14); this trend was statistically significant (p = 0.006). Side scatter (SSC), a measure of cellular granularity was also elevated in AD patients (p = 0.02). The elevated expression of human leukocyte antigen (HLA-DR) was not found to be significant between age-matched controls and AD patients. When conditioned media from M/M phi from five AD and two control patients were evaluated for neurotoxicity, three of the five culture supernatants from AD patients induced apoptosis in neural cell aggregate cultures. Electrophoretic mobility shift assays revealed that these three supernatants also triggered NF-kappaB translocation to the nucleus. Surprisingly, in vitro neurotoxicity was induced by M/M phi supernatants having a lower percentage of CD14+/CD69+ cells. Elevation of the CD14+/CD69+ subset in AD patients may therefore represent a manifestation in the peripheral blood of the pathological events occurring in the brain but may not be directly involved in neural cell toxicity.

Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains.

For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of transformed calli (77%) when used in conjunction with tobacco nurse culture during four days of cocultivation. Using this strain, kanamycin (76%) and hygromycin (77%) were equally effective selective agents, but for strain LBA4404(pBI1042) percentage of transformed calli was higher for hygromycin (63%) than for kanamycin (17%). The procedures and strains shown to be optimal for transformation of pea callus will now be complemented by a pea regeneration system.

Molecular characterization of the virC genes of the Ti plasmid.

The virC (formerly bak) complementation group of the nopaline-type Ti plasmid pTiC58 encodes two proteins, VirC1 and VirC2. According to the primary structure of the polypeptides predicted by the nucleotide sequence, VirC1 is composed of 231 amino acids with a total molecular mass of 25.5 kilodaltons, and VirC2 is composed of 202 amino acids with a molecular mass of 22.1 kilodaltons. The pTiC58 VirC1 and VirC2 polypeptides are equal in length to VirC1 and VirC2 of the octopine-type plasmid pTiA6NC. VirC1 proteins of pTiC58 and pTiA6NC are identical at 202 (87.4%) of the amino acid residues, and this homology is distributed fairly evenly throughout the protein. VirC2 identities occur at 142 residues (70.3%), but fall predominantly into two blocks of higher homology (84.6 and 78.5%) separated by a 41-residue segment of much lower homology (29.3%). Mutations in virC resulted in attenuated virulence on all hosts tested, the severity of attenuation varying markedly depending on the type of plant inoculated. For example, the attenuation was more pronounced on Kalanchoe than on sunflower or jimson weed. Virulence was restored to normal on all hosts by in-trans complementation with corresponding nonmutant DNA fragments of pTiC58 or of the octopine-type plasmid pTi15955. Two oligopeptides from within the predicted pTiC58 VirC1 polypeptide were synthesized and used to raise antibodies. These antibodies were used to detect the VirC1 product of both pTiC58 and pTi15955. In both cases, virC was expressed constitutively in the Agrobacterium tumefaciens ros mutant. The homology between virC genes of octopine- and nopaline-type Ti plasmids thus includes a conservation of genetic regulatory control mechanisms as well as considerable conservation of the primary structure of the protein products.

The aminoglycoside-resistance operon of the plasmid pSa: nucleotide sequence of the streptomycin-spectinomycin resistance gene.

The nucleotide sequence of the probable C terminus of the kanamycin-resistance gene (KmR) and the probable complete sequence of the streptomycin-spectinomycin-resistance gene (SpR) of the IncW plasmid pSa have been determined. The two genes appear to be oriented in the same direction and separated by a spacer region of 53 bp, with transcription proceeding from the KmR gene into the SpR gene. An RNA transcript encompassing the C terminus of the KmR gene, the 53-base spacer, and the N terminus of the SpR gene has the potential to form a stem-loop structure with a free energy value of -68 kcal/mol. The SpR gene of pSa has extensive sequence homology with the aadA gene of the plasmid R538-1. Comparison of the proposed amino acid sequence of the KmR protein of pSa with those of two aminoglycoside phosphotransferases revealed a region of potential homology with those proteins.

Physical properties of fluorosis bone.

The bones of two patients, one with a moderate and one with a severe chronic industrial fluorosis (stage I-II and stage III), and the bones of three control persons were examined. The following parameters were determined: the fracture load, the fracture load/unit area (resistance to pressure) of the body of the first lumbar vertebra, the bending strength of the neck of the femur and of the lower third of the femur, the fracture load/unit area and the modulus of elasticity of femoral slices 2 cm thick and of precisely defined cylinders from the femoral cortex. The microhardness according to Vickers on the cross section of the femur was also determined. The results obtained are discussed with regard to fluoride therapy of osteoporosis.