Richard E. Isaacson

In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection.

Azithromycin, a novel azalide antibiotic, concentrated in human and mouse polymorphonuclear leukocytes (PMNs), murine peritoneal macrophages, and mouse and rat alveolar macrophages, attaining intracellular concentrations up to 226 times the external concentration in vitro. In murine peritoneal macrophages, azithromycin achieved concentration gradients (internal to external) up to 26 times higher than erythromycin. The cellular uptake of azithromycin was dependent on temperature, viability, and pH and was decreased by 2,4-dinitrophenol. Azithromycin did not decrease phagocyte-mediated bactericidal activity or affect PMN or macrophage oxidative burst activity (H2O2 release or Nitro Blue Tetrazolium reduction, respectively). Azithromycin remained in cells for several hours, even after extracellular drug was removed. However, its release was significantly enhanced by phagocytosis of Staphylococcus aureus (82 versus 23% by 1.5 h). In vivo, 0.05 micrograms of azithromycin was found in peritoneal fluids of mice 20 h after oral treatment with a dose of 50 mg/kg. Following caseinate-induced PMN infiltration, there was a sixfold increase in peritoneal cavity azithromycin to 0.32 micrograms, most of which was intracellular. Therefore, the uptake, transport, and later release of azithromycin by these cells demonstrate that phagocytes may deliver active drug to sites of infection.

Longitudinal investigation of the age-related bacterial diversity in the feces of commercial pigs.

The importance of bacteria in the gastrointestinal tracts of animals is widely acknowledged as important. However, very little is known about composition and distribution of the microbial population in lower intestinal tracts of animals. Because most bacterial species in pig intestines have not been cultured, it has been difficult to analyze bacterial diversity by conventional culture methods. Even with the development of culture independent 16S rRNA gene sequencing, the previous methods were slow and labor intensive. Therefore, high throughput pyrosequencing of 16S rDNA libraries was used in this study in order to explore the bacterial diversity of the pig feces. In our two trials, fecal samples from individual pigs were collected five times at 3-week intervals, and the 16S rRNA genes in the community DNAs from fecal samples were sequenced and analyzed. This longitudinal study design identified that microbial populations in the feces of the each pig continued to change as pigs aged. The variations of bacterial diversity of the animals were affected by less abundant bacterial components of the feces. These results help us to understand the age-related bacterial diversity in the commercial pig feces.

Modulations of the Chicken Cecal Microbiome and Metagenome in Response to Anticoccidial and Growth Promoter Treatment

With increasing pressures to reduce or eliminate the use of antimicrobials for growth promotion purposes in production animals, there is a growing need to better understand the effects elicited by these agents in order to identify alternative approaches that might be used to maintain animal health. Antibiotic usage at subtherapeutic levels is postulated to confer a number of modulations in the microbes within the gut that ultimately result in growth promotion and reduced occurrence of disease. This study examined the effects of the coccidiostat monensin and the growth promoters virginiamycin and tylosin on the broiler chicken cecal microbiome and metagenome. Using a longitudinal design, cecal contents of commercial chickens were extracted and examined using 16S rRNA and total DNA shotgun metagenomic pyrosequencing. A number of genus-level enrichments and depletions were observed in response to monensin alone, or monensin in combination with virginiamycin or tylosin. Of note, monensin effects included depletions of Roseburia, Lactobacillus and Enterococcus, and enrichments in Coprococcus and Anaerofilum. The most notable effect observed in the monensin/virginiamycin and monensin/tylosin treatments, but not in the monensin-alone treatments, was enrichments in Escherichia coli. Analysis of the metagenomic dataset identified enrichments in transport system genes, type I fimbrial genes, and type IV conjugative secretion system genes. No significant differences were observed with regard to antimicrobial resistance gene counts. Overall, this study provides a more comprehensive glimpse of the chicken cecum microbial community, the modulations of this community in response to growth promoters, and targets for future efforts to mimic these effects using alternative approaches.

Microbial shifts in the swine distal gut in response to the treatment with antimicrobial growth promoter, tylosin

Antimicrobials have been used extensively as growth promoters (AGPs) in agricultural animal production. However, the specific mechanism of action for AGPs has not yet been determined. The work presented here was to determine and characterize the microbiome of pigs receiving one AGP, tylosin, compared with untreated pigs. We hypothesized that AGPs exerted their growth promoting effect by altering gut microbial population composition. We determined the fecal microbiome of pigs receiving tylosin compared with untreated pigs using pyrosequencing of 16S rRNA gene libraries. The data showed microbial population shifts representing both microbial succession and changes in response to the use of tylosin. Quantitative and qualitative analyses of sequences showed that tylosin caused microbial population shifts in both abundant and less abundant species. Our results established a baseline upon which mechanisms of AGPs in regulation of health and growth of animals can be investigated. Furthermore, the data will aid in the identification of alternative strategies to improve animal health and consequently production.

Transmission of Salmonella typhimurium to swine.

These studies were designed to determine the rate of transmission and the colonization pattern of Salmonella typhimurium in swine. Two experiments were conducted. In experiment 1, swine challenged per os with either S. typhimurium strain 798T + or strain 798N + were exposed to heterologous feces. Following exposure to heterologous strains, heterologous Salmonella were recovered from the feces of infected swine within 3 days and from the tonsil and ileum at necropsy. Bacterial populations in swine initially challenged with Salmonella remained constant. In experiment 2, Salmonella-free swine were commingled with a population of pigs that were shedding 2.69 log10 CFU Salmonella/gram feces. Salmonella was recovered from pooled fecal samples from the commingled swine on day 2 post-exposure to the infected group. Low numbers of Salmonella were detected in the ileocolic lymph node, ileum, cecum or spleen of all commingled swine throughout the necropsy period. These data provide a means for evaluating transmission of Salmonella to a population of swine which may be used to study the mechanisms involved in transmission and maintenance of the disease.

The intestinal microbiome of the pig

Abstract The intestinal microbiome has been the subject of study for many decades because of its importance in the health and well being of animals. The bacterial components of the intestinal microbiome have closely evolved as animals have and in so doing contribute to the overall development and metabolic needs of the animal. The microbiome of the pig has been the subject of many investigations using culture-dependent methods and more recently using culture-independent techniques. A review of the literature is consistent with many of the ecologic principles put forth by Rene Dubos. Animals develop an intestinal microbiome over time and space. During the growth and development of the pig, the microbiome changes in composition in a process known as the microbial succession. There are clear and distinct differences in the composition of the pig intestinal microbiome moving from the proximal end of the intestinal tract to the distal end. The majority (>90%) of the bacteria in the pig intestinal microbiome are from two Phyla: Firmicutes and Bacteroidetes. However, the ileum has a high percentage of bacteria in the phylum Proteobacterium (up to 40%). Perturbations to the microbiome occur in response to many factors including stresses, treatment with antibiotics, and diet.

Type 1 Fimbriae of Salmonella enterica Serovar Typhimurium Bind to Enterocytes and Contribute to Colonization of Swine In Vivo

ABSTRACT Salmonella enterica serovar Typhimurium strain 798 is a clinical isolate from a pig and is known to be able to cause persistent, asymptomatic infections. This strain also is known to exist in two phenotypes (adhesive and nonadhesive to enterocytes) and can switch between the two phenotypes at a rate consistent with phase variation. Cells in the adhesive phenotype are more readily phagocytosed by leukocytes than nonadhesive cells. Once in a leukocyte, adhesive-phase cells survive while nonadhesive-phase cells die. In the present study, nonadhesive mutants were obtained with the transposon TnphoA. A nonadhesive mutant was selected for study and was shown by electron microscopy not to produce fimbriae. The gene encoding the adhesin was cloned and sequenced. Based on its sequence, the adhesin was shown to be FimA, the major subunit of type 1 fimbriae. The nonadhesive mutant was attenuated in its ability to colonize both mouse and pig intestines, but remained capable of systemic spread in mice. The nonadhesive mutant was phagocytosed to the same extent as parental cells in the adhesive phase and then survived intracellularly. These results demonstrated that type 1 fimbriae were important for attachment to enterocytes and promoted intestinal colonization. However, they were not important in promoting phagocytosis or intracellular survival.

Distribution of Salmonella in Swine Production Ecosystems

The objective of this 2-year field survey was to sample multiple ecological compartments within swine production systems to identify potential sources of Salmonella infection for swine. Twelve single-site production systems within Illinois were identified by slaughter sampling to have detectable Salmonella in swine and therefore selected for study. There were four visits to each farm during a 5-month period. Fecal samples were obtained from swine and other wild and domestic mammals. Arthropods and environmental samples of feed, water, pen floors, boots, and bird feces were also collected. All 8,066 samples obtained were cultured to detect Salmonella. Salmonella was detected on 11 of the 12 farms. There were 206 positive cultures, including samples from swine (83), pen floors (54), boots (32), flies (16), mice (9), cats (3), and birds (3). Swine shedding Salmonella in feces were detected on 9 of the 12 farms. The more Salmonella-abundant ecological compartments were cats (12% of samples positive), boots (11%), bird feces (8%), flies (6%), and mice (5%); 2.1% of 4,024 swine samples were positive. All 221 feed samples were negative for Salmonella. There was a correlation between a farm having a high prevalence of shedding Salmonella in pigs and a high abundance on pen floors, flies, and boots. The most common serotypes detected were Derby, Agona, Worthington, and Uganda, which were distributed throughout the ecosystem, suggesting widespread transmission across ecological compartments. The ubiquitous distribution of Salmonella suggests that an effective control strategy must target multiple compartments of the swine production ecosystem.

The pig gut microbial diversity: Understanding the pig gut microbial ecology through the next generation high throughput sequencing

The importance of the gut microbiota of animals is widely acknowledged because of its pivotal roles in the health and well being of animals. The genetic diversity of the gut microbiota contributes to the overall development and metabolic needs of the animal, and provides the host with many beneficial functions including production of volatile fatty acids, re-cycling of bile salts, production of vitamin K, cellulose digestion, and development of immune system. Thus the intestinal microbiota of animals has been the subject of study for many decades. Although most of the older studies have used culture dependent methods, the recent advent of high throughput sequencing of 16S rRNA genes has facilitated in depth studies exploring microbial populations and their dynamics in the animal gut. These culture independent DNA based studies generate large amounts of data and as a result contribute to a more detailed understanding of the microbiota dynamics in the gut and the ecology of the microbial populations. Of equal importance, is being able to identify and quantify microbes that are difficult to grow or that have not been grown in the laboratory. Interpreting the data obtained from this type of study requires using basic principles of microbial diversity to understand importance of the composition of microbial populations. In this review, we summarize the literature on culture independent studies of the pig gut microbiota with an emphasis on its succession and alterations caused by diverse factors.

Do microbial interactions and cultivation media decrease the accuracy of Salmonella surveillance systems and outbreak investigations

Cultivation methods are commonly used in Salmonella surveillance systems and outbreak investigations, and consequently, conclusions about Salmonella evolution and transmission are highly dependent on the performance characteristics of these methods. Past studies have shown that Salmonella serotypes can exhibit different growth characteristics in the same enrichment and selective media. This could lead not only to biased conclusions about the dominant strain present in a sample with mixed Salmonella populations, but also to a low sensitivity for detecting a Salmonella strain in a sample with only a single strain present. The objective of this study was to determine whether cultivation media select preferentially for specific strains of Salmonella in heterogeneous cultures. In this study, four different Salmonella strains (one Salmonella Newport, two Salmonella Typhimurium, and one Salmonella Enteritidis) were competed in a broth-based experiment and a bovine fecal experiment with varied combinations and concentrations of each strain. In all experiments, the strain of Salmonella Newport was the most competitive, regardless of the starting concentration and cultivation protocol. One strain of Salmonella Typhimurium was rarely detected in competition, even when it was the only strain present in bovine feces. Overall, the probability of detecting a specific Salmonella strain had little to do with its starting concentration in the sample. The bias introduced by culture could be dramatically biasing Salmonella surveillance systems and hindering traceback investigations during Salmonella outbreaks. Future studies should focus on the microbiological explanations for this Salmonella interstrain variability, approaches for minimizing the bias, and estimations of the public health significance of this bias.