Antimicrobial activity of lupulone against Clostridium perfringens in the chicken intestinal tract jejunum and caecum.

OBJECTIVES: Owing to the spread of antibiotic resistance among human infectious agents, there is a need to research antibiotic alternatives for use in animal agricultural systems. Antibiotic-free broiler chicken production systems are known to suffer from frequent outbreaks of necrotic enteritis due in part to pathogenic type A Clostridium perfringens. Hop (Humulus lupulus) bitter acids are known to possess potent antimicrobial activity. Lupulone was evaluated for in vivo antimicrobial activity to inhibit C. perfringens in a chick gastrointestinal colonization model. METHODS: Using a week-2 per os inoculated C. perfringens chicken colonization model, C. perfringens counts in mid-intestinal and caecal contents were compared between chickens administered lupulone at 62.5, 125 and 250 ppm in drinking water versus 0 ppm control. Results At day 22, post-hatch intestinal C. perfringens counts of lupulone-treated chickens were significantly lower (P < 0.05) than water-treated control groups in both jejunal and caecal sampling sites across all lupulone dosages tested. CONCLUSIONS: Lupulone administered through water inhibits gastrointestinal levels of inoculated pathogenic clostridia within the chicken gastrointestinal tract. Lupulone was effective within the chemically complex mixture of material within the gastrointestinal tract, thereby making this agent a target of further research as an antibiotic alternative for this and possibly other intestinal infections.

Quantitative analysis of the intestinal bacterial community in one- to three-week-old commercially reared broiler chickens fed conventional or antibiotic-free vegetable-based diets.

AIMS: To explore the effect of drug-free poultry production on the intestinal microflora of broiler chickens, the bacterial community of this environment was quantitatively profiled in both conventionally reared birds and birds reared without antibiotic growth promotants (AGPs) on a vegetable-based diet. METHODS AND RESULTS: Quantitative, real-time PCR with group-specific 16S rDNA primer sets was used to enumerate the abundance of the following chicken gastrointestinal (GI) tract phylogenetic groups: the Clostridium leptum-Faecalibacterium prausnitzii subgroup (Clostridium genus cluster IV), the Clostridium coccoides - Eubacterium rectale subgroup (Clostridium cluster XIVa and XIVb), the Bacteroides group (including Prevotella and Porphyromonas), Bifidobacterium spp., the Enterobacteriaceae, the Lactobacillus group (including the genera Leuconostoc, Pediococcus, Aerococcus and Weissella), the Clostridium perfringens subgroup (Clostridium cluster I), Enterococcus spp., Veillonella spp., Atopobium spp., Campylobacter spp. and the domain Bacteria. A species-specific 5'-nuclease (Taqman) assay was also employed to specifically assess Cl. perfringens abundance. Ten birds were sampled from each of two commercial chicken houses, one in which feed was supplemented with AGPs and exogenous animal protein, and the other vegetable-based and drug-free, at 7, 14 and 21 days of age. The ileal community was dominated by two large populations, the lactobacilli and the Enterobacteriaceae, with those taxa much more numerous in drug-free vegetable-based diet fed birds than those conventionally reared at the 7- and 14-day time periods. The progressive changes in microflora in both the conventional and drug-free caeca were similar to each other, with the Enterobacteriaceae sequences dominating at day 7, but being replaced by obligate anaerobe signature sequences by day 14. Of note was the finding that all the day 14 and day 21 replicate caecal samples from the drug-free house were positive for Campylobacter spp. averaging >10(8) 16S rDNA gene copies per gram wet weight. CONCLUSIONS: Quantitative, real-time PCR indicates that the effects of drug-free rearing on the chicken GI tract microbial community are most pronounced in the ileal region, but AGPs may be important in controlling Campylobacter colonization of the caecum. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative taxonomic understanding of the shifting microbial ecology of the broiler chicken gut microbiota is important in the light of AGP withdrawal. AGP withdrawal has occurred in response to concerns over the transfer of antimicrobial-resistant bacteria to humans via the food production chain.

Molecular subtyping of poultry-associated type A Clostridium perfringens isolates by repetitive-element PCR.

Clostridium perfringens strains (type A) isolated from an integrated poultry operation were subtyped using repetitive-element PCR with Dt primers. Isolates were obtained from fecal, egg shell, fluff, and carcass rinse samples as part of a previously reported temporally linked epidemiological survey. A total of 48 isolates of C. perfringens were obtained from different stages of the broiler chicken production chain from two separate breeder farms that supplied a single hatchery that in turn provided chicks to a single grow-out farm whose flocks were processed at a single plant. All 48 isolates were typeable (100% typeability) by repetitive-element PCR with Dt primers. This subtyping method was highly reproducible and discriminatory. By repetitive-element PCR with Dt primers, isolates were classified into four major branches with 12 subgroups or clades. The Simpson's index of discrimination was calculated to be 0.96 for groupings of >95% correlation. Toxin gene profiles of the isolates indicated that all of the isolates were C. perfringens alpha-toxin gene positive and 46 of 48 isolates were beta2-toxin gene positive. All strains were negative for beta- and epsilon-toxin genes. Repetitive sequence-based PCR was found to be a technically practical and reproducible means of subtyping C. perfringens libraries from specific epidemiological or production environment settings.

Methylosarcina fibrata gen. nov., sp. nov. and Methylosarcina quisquiliarum sp.nov., novel type 1 methanotrophs.

Two novel species of obligate methane-oxidizing bacteria, isolated from landfill soil, were characterized. Both strains were unusual in that some members of the population grew in irregularly shaped, refractile cell packets that resembled sarcina-like clusters. Electron microscopy revealed that the cell packets were covered with a slime layer and the cells contained many large granular inclusion bodies. The individual cells of each strain were sometimes motile and had differing morphologies. Isolate AML-C10T was always coccoidal in shape, and the cells were covered with extracellular fibrils. Isolate AML-D4T was pleomorphic, changing from rod to coccal form, sometimes exhibiting an unusual fusiform morphology. AML-D4T lacked the extensive fibrillar matrix observed with AML-C10T. Both strains utilized only methane and methanol as carbon sources. In stationary phase, the cells of each strain swelled in size and formed cysts. Aside from morphological differences, strains could also be distinguished from each other by cellular protein patterns, as well as by temperature and pH tolerances. 16S rDNA phylogenetic analysis showed that these are type I methanotrophs (family: Methylococcaceae) most closely related to the Methylobacter/Methylomicrobium clade, although they form a monophyletic grouping supported by moderately high bootstrap values. By 16S rDNA database searches, the most similar species to both isolates were Methylobacter spp. However, partial particulate methane monooxygenase sequence analysis suggested that these bacteria might be more closely related to Methylomicrobium than Methylobacter. Furthermore, cellular fatty acid profiles of the strains more closely resemble those of Methylomicrobium, although the absence of significant levels of 16:1omega5c argues for the uniqueness of these two strains. On the basis of the results described here, it is proposed that a new genus should be created, Methylosarcina gen. nov., harbouring two species, Methylosarcina fibrata sp. nov. (type species) and Methylosarcina quisquiliarum sp. nov. The type strains are AML-C10T (= ATCC 700909T = DSM 13736T) and AML-D4T (= ATCC 700908T = DSM 13737T), respectively.

Methanotroph diversity in landfill soil: isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis.

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4. 8 versus 6.8), air/CH(4)/CO(2) headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1x nitrate minimal salts [NMS] versus 0.2x NMS). Screening of the isolates showed that the nutrient-rich 1x NMS selected for type I methanotrophs, while the nutrient-poor 0.2x NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobium group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published Methylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.

Anxiety and neurological disorders.

Anxiety disorders occur quite frequently in patients who have neurologic disorders. In fact, several studies show that anxiety disorders occur more frequently than mood disorders in patients with neurologic conditions. Despite high prevalence rates, investigators have neglected the study of anxiety disorders in these patients. Two major reasons for the neglect are the diagnostic hierarchy and exclusion criterion found in Diagnostic and Statistical Manual (DSM)-III-R and DSM-IV. This article discusses problems in the diagnosis of anxiety disorders in patients with neurologic conditions and presents a brief literature review of the relationship between anxiety and neurologic disorders. In addition, the authors describe the differential diagnosis of anxiety in neurologic conditions and treatment options.

Factitious disorders.

Reports of factitious disorders, Munchausen's syndrome, and self-induced illness exist throughout medical history. In practice, disease simulation represents a spectrum of behaviors that range from relatively common and benign (e.g., pleading illness to avoid an unwanted social obligation) to rare and malignant forms (e.g., Munchausen's syndrome and factitious disorder by proxy). Factitious disorders are differentiated from malingering by the goal that motivates the individual's behavior. The only apparent goal in factitious illness is to gain the sick role; the goal in malingering is to gain rewards, such as compensation, or to avoid the unwanted, such as military service or jail. This article summarizes clinically relevant information on factitious disorders for primary care physicians.

Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.

Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project's taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type.

A treatment algorithm for the management of anxiety in primary care practice.

Patients frequently present to primary care physicians with somatic symptoms that mask an underlying anxiety disorder. As a result, unnecessary diagnostic tests are ordered, and inappropriate medications are prescribed. Psychiatrists may help improve their primary care colleagues' ability to identify and treat these anxiety disorders. This paper reviews the adverse effects of untreated anxiety in managed care settings and outlines a treatment algorithm that psychiatrists may wish to use to assist primary care physicians in the cost-efficient, pharmacologic treatment of anxiety disorders in their patients.

Temporal Variation in Genetic Diversity and Structure of a Lotic Population of Burkholderia (Pseudomonas) cepacia.

The genetic structure and temporal patterns of genetic diversity in a population of Burkholderia (Pseudomonas) cepacia, isolated from a southeastern blackwater stream, were investigated by multilocus enzyme electrophoresis. Allelic variation in seven structural gene loci was monitored at a single stream location at 0, 6, 12, and 24 h and at 2, 4, 8, 16, and 32 days. Over the length of the study, 217 isolates were collected, from which 65 unique electrophoretic types (ETs) were identified. Most of these ETs were present at only one or two time periods and were considered transients; however, one resident ET was particularly abundant (64 of the 217 isolates [29.4%]) and was found at all time points except day 32. The mean genetic diversity of the entire population was 0.520, and the index of association (a measure of multilocus linkage disequilibrium) was 1.33. These results, taken in conjunction with a previous study focusing on spatial patterns of genetic diversity in lotic B. cepacia, show that these bacterial populations exhibit greater variability among sites than within a site over time, suggesting relative stability over short time periods.

Genetic structure of a lotic population of Burkolderia (Pseudomonas) cepacia.

The genetic structure of a population of Burkholderia (Pseudomonas) cepacia isolated from a southeastern blackwater stream was investigated by using multilocus enzyme electrophoresis to examine the allelic variation in eight structural gene loci. Overall, 213 isolates were collected at transect points along the stream continuum, from both the sediments along the bank and the water column. Multilocus enzyme electrophoresis analysis revealed 164 distinct electrophoretic types, and the mean genetic diversity of the entire population was 0.574. Genetic diversity values did not vary spatially along the stream continuum. From a canonical discriminant analysis, Mahalonobis distances (measurements of genetic similarity between populations) revealed significant differences among the subpopulations at the sediment sampling points, suggesting bacterial adaptation to a heterogeneous (or patchy) microgeographical environment. Multilocus linkage disequilibrium analysis of the isolates revealed only limited association between alleles, suggesting frequent recombination, relative to binary fission, in this population. Furthermore, the dendrogram created from the data of this study and the allele mismatch distribution are typical of a population characterized by extensive genetic mixing. We suggest that B. cepacia be added to the growing list of bacteria that are not obligatorily clonal.

The clinical and financial burden of mood disorders. Cost and outcome.

Depressive disorders are a chronic, recurrent, and severe burden to both patients and their families. Depressive disorders represent a major national public health problem, ranking within the top 10 most costly diseases in the United States. In 1990, depressive disorders afflicted at least 11 million Americans and cost the U.S. economy an estimated $44 billion. In addition, affective disorders are associated with increased accident rates, increased rates of substance abuse (especially alcoholism), increased medical hospitalization, and an increase in somatic illnesses and outpatient medical utilization. Despite their ranking as a major health problem, depressive disorders are often underdiagnosed and undertreated. Brief treatment strategies that focus only on acute episodes are often ineffective and result in chronic impairment, impairing performance at work and socially. Inadequate treatment increases costs, suffering, and lost productivity. Recent data suggest that effective treatment of depression requires long-term, skillful follow-up and active pharmacotherapy.

A combined treatment approach to anxiety in the medically ill.

Anxiety occurs frequently in patients who are medically ill. A proper search for the underlying cause of the anxiety is essential if the clinician is to make a correct diagnosis and initiate appropriate treatment. Two aspects of the patient's history are particularly important during assessment: the duration and severity of medical illness (and treatments) and the duration and severity of anxiety symptoms. When acute anxiety is encountered, the clinician must rule out a rapidly worsening medical condition, substance-induced anxiety (toxicity or withdrawal), and a psychological reaction to stressors associated with the medical illness. During evaluation of medically ill individuals with chronic anxiety, the clinician must rule out medical disorders that can mimic anxiety disorders, psychiatric disorders associated with anxiety symptoms, and poor adjustment to the medical illness. It is also worth remembering that anxiety disorders occur at an increased frequency in individuals who have chronic medical illness.

Diagnostic considerations and treatment approaches to underlying anxiety in the medically ill.

Autonomic and somatic manifestations of anxiety are common in medically ill patients. When anxiety symptoms occur in such patients, the psychiatrist's ability to rapidly identify anxiety and perform a proper differential diagnosis is important, and can be life-saving. For example, a patient with deteriorating cardiac or pulmonary function may be misidentified as primarily "anxious" and referred to the psychiatrist. The change in the patient's medical status is missed until the situation becomes critical. Medication side effects can also cause anxiety symptoms in the medically ill patient. It is important for the psychiatrist to have knowledge of medications commonly associated with such side effects. Diagnosing primary anxiety disorders, such as generalized anxiety disorder, panic disorder, or posttraumatic stress disorder, can be difficult in medically ill patients, but it is an important skill for the consulting psychiatrist. Anxiety can occur secondary to the stress or fear associated with illness, particularly serious illness. This presentation will discuss all of the aforementioned aspects of anxiety in the patient with concomitant medical illness.

Psychopharmacology in the elderly.

By the year 2030, it is estimated that 17% of the population (52 million people) will be over age 65. Most of these individuals will be taking several medications, and one or more of these medications may be a psychotropic, that is, an antidepressant, anxiolytic, antipsychotic, or lithium. This article summarizes the pharmacokinetic changes that accompany aging and highlight how these changes impact the elderly.

Psychiatric disorders in the medically ill.

Psychiatric disorders commonly coexist in patients with medical illness. Three disorders encountered frequently are anxiety disorders, depression, and delirium. The authors discuss these psychiatric disorders and their treatment in medically ill patients. Psychiatric consultation may be helpful in such cases.

Anxiety and mood disorders in medically ill patients.

In medically ill patients, anxiety symptoms and mood alteration are common. Anxiety or mood alteration may be due to a reaction to the stress of illness, a preexisting psychiatric disorder, a manifestation of the medical condition, or an adverse effect of medication. Psychiatrists are frequently called upon to differentiate among these causes and to recommend treatment. This article reviews anxiety, depression, and mania, which are frequently associated with physical disease, and provides an approach to differential diagnosis and treatment.