Antibiotics in the aquatic environment - A review - Part II

The human intestinal microbiota is essential to the health of the host and plays a role in nutrition, development, metabolism, pathogen resistance, and regulation of immune responses. Antibiotics may disrupt these coevolved interactions, leading to acute or chronic disease in some individuals. Our understanding of antibiotic-associated disturbance of the microbiota has been limited by the poor sensitivity, inadequate resolution, and significant cost of current research methods. The use of pyrosequencing technology to generate large numbers of 16S rDNA sequence tags circumvents these limitations and has been shown to reveal previously unexplored aspects of the “rare biosphere.” We investigated the distal gut bacterial communities of three healthy humans before and after treatment with ciprofloxacin, obtaining more than 7,000 full-length rRNA sequences and over 900,000 pyrosequencing reads from two hypervariable regions of the rRNA gene. A companion paper in PLoS Genetics (see Huse et al., doi: 10.1371/journal.pgen.1000255) shows that the taxonomic information obtained with these methods is concordant. Pyrosequencing of the V6 and V3 variable regions identified 3,300–5,700 taxa that collectively accounted for over 99% of the variable region sequence tags that could be obtained from these samples. Ciprofloxacin treatment influenced the abundance of about a third of the bacterial taxa in the gut, decreasing the taxonomic richness, diversity, and evenness of the community. However, the magnitude of this effect varied among individuals, and some taxa showed interindividual variation in the response to ciprofloxacin. While differences of community composition between individuals were the largest source of variability between samples, we found that two unrelated individuals shared a surprising degree of community similarity. In all three individuals, the taxonomic composition of the community closely resembled its pretreatment state by 4 weeks after the end of treatment, but several taxa failed to recover within 6 months. These pervasive effects of ciprofloxacin on community composition contrast with the reports by participants of normal intestinal function and with prior assumptions of only modest effects of ciprofloxacin on the intestinal microbiota. These observations support the hypothesis of functional redundancy in the human gut microbiota. The rapid return to the pretreatment community composition is indicative of factors promoting community resilience, the nature of which deserves future investigation.

/pdf/the-pervasive-effects-of-an-antibiotic-on-the-human-gut-5c30xb8367.pdf

The Pervasive Effects of an Antibiotic on the Human Gut Microbiota, as Revealed by Deep 16S rRNA Sequencing

This study explores antibiotic resistance genes (ARGs) as emerging environmental contaminants. The purpose of this study was to investigate the occurrence of ARGs in various environmental compartments in northern Colorado, including Cache La Poudre (Poudre) River sediments, irrigation ditches, dairy lagoons, and the effluents of wastewater recycling and drinking water treatment plants. Additionally, ARG concentrations in the Poudre River sediments were analyzed at three time points at five sites with varying levels of urban/agricultural impact and compared with two previously published time points. It was expected that ARG concentrations would be significantly higher in environments directly impacted by urban/agricultural activity than in pristine and lesser-impacted environments. Polymerase chain reaction (PCR) detection assays were applied to detect the presence/absence of several tetracycline and sulfonamide ARGs. Quantitative real-time PCR was used to further quantify two tetracycline ARGs (tet(W) and...

Antibiotic Resistance Genes as Emerging Contaminants: Studies in Northern Colorado†

Altering the Intestinal Microbiota during a Critical Developmental Window Has Lasting Metabolic Consequences

The connection between the gut microbiota and the aetiology of obesity and cardiometabolic disorders is increasingly being recognized by clinicians. Our gut microbiota might affect the cardiometabolic phenotype by fermenting indigestible dietary components and thereby producing short-chain fatty acids (SCFA). These SCFA are not only of importance in gut health and as signalling molecules, but might also enter the systemic circulation and directly affect metabolism or the function of peripheral tissues. In this Review, we discuss the effects of three SCFA (acetate, propionate and butyrate) on energy homeostasis and metabolism, as well as how these SCFA can beneficially modulate adipose tissue, skeletal muscle and liver tissue function. As a result, these SCFA contribute to improved glucose homeostasis and insulin sensitivity. Furthermore, we also summarize the increasing evidence for a potential role of SCFA as metabolic targets to prevent and counteract obesity and its associated disorders in glucose metabolism and insulin resistance. However, most data are derived from animal and in vitro studies, and consequently the importance of SCFA and differential SCFA availability in human energy and substrate metabolism remains to be fully established. Well-controlled human intervention studies investigating the role of SCFA on cardiometabolic health are, therefore, eagerly awaited.

Short-chain Fatty Acids in Control of Body Weight and Insulin Sensitivity

Recent concerns about the use of growth-promoting antibiotics in pig diets have renewed interest in the immunologic and growth-regulating functions of the gastrointestinal (GI) tract. The numerically dense and metabolically active microbiota ofthe pig GI tract represents a key focal point for such questions. The intestinal microbiota is viewed typically as a beneficial entity for the host. Intestinal bacteria provide both nutritional and defensive functions for their host. However, the host animal invests substantially in defensive efforts to first sequester gut microbes away from the epithelial surface, and second to quickly mount immune responses against those organisms that breach epithelial defenses. The impact of host responses to gut bacteria and their metabolic activities require special consideration when viewed in the context of pig production in which efficiency of animal growth is a primary objective. Here, we summarize the working hypothesis that antibiotics improve the efficiency of animal growth via their inhibition of the normal microbiota, leading to increased nutrient utilization and a reduction in the maintenance costs ofthe GI system. In addition, novel molecular ecology techniques are described that can serve as tools to uncover the relationship between intestinal microbiology and growth efficiency.

Antibiotics as growth promotants:mode of action

The gastrointestinal (GI) microbiota of mammals is characterized by its high population density, wide diversity and complexity of interactions. While all major groups of microbes are represented, bacteria predominate. Importantly, bacterial cells outnumber animal (host) cells by a factor of ten and have a profound influence on nutritional, physiological and immunological processes in the host animal. Our knowledge of the molecular and cellular bases of host-microbe interactions is limited, though critically needed to determine if and how the GI microbiota contributes to various enteric disorders in humans and animals. Traditionally, GI bacteria have been studied via cultivation-based techniques, which are labor intensive and require previous knowledge of individual nutritional and growth requirements. Recently, findings from culture-based methods have been supplemented with molecular ecology techniques that are based on the 16S rRNA gene. These techniques enable characterization and quantification of the microbiota, while also providing a classification scheme to predict phylogenetic relationships. The choice of a particular molecular-based approach depends on the questions being addressed. Clone libraries can be sequenced to identify the composition of the microbiota, often to the species level. Microbial community structure can be analyzed via fingerprinting techniques, while dot blot hybridization or fluorescent in situ hybridization can measure abundance of particular taxa. Emerging approaches, such as those based on functional genes and their expression and the combined use of stable isotopes and biomarkers, are being developed and optimized to study metabolic activities of groups or individual organisms in situ. Here, a critical summary is provided of current molecular ecological approaches for studying the GI microbiota.

/pdf/molecular-ecological-analysis-of-the-gastrointestinal-3vwlqmystq.pdf

Molecular ecological analysis of the gastrointestinal microbiota: a review.

Cultivation-independent microbial molecular ecology approaches were used to examine the effects of antibiotic growth promoters on the pig ileal microbiota. Five-week-old barrows were fitted with a simple T-cannula at the distal ileum. Three diets meeting or exceeding the minimum nutrient requirements were fed for 5 wk and supplemented as follows: 1) negative control (no antibiotic; n = 5), 2) continuous tylosin administration (n = 5), and 3) an antibiotic rotation sequence (wk 1, chlorotetracycline sulfathiazole penicillin; wk 2, bacitracin and roxarsone; wk 3, lincomycin; wk 4, carbadox; wk 5, virginiamycin; n = 5). Ileal luminal contents were collected for DNA isolation at the end of each of the 5 wk of the testing period. The V3 region of 16S rDNA was amplified by PCR and analyzed via denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction (qPCR). Resulting PCR-DGGE band numbers (bacterial species) were counted, and the banding patterns analyzed by calculating Sorenson's pairwise similarity coefficients (C(S)), an index measuring bacterial species in common among samples. Band numbers and total bacterial DNA concentrations decreased (P < 0.05) temporally in antibiotic-treated pigs compared with controls. Comparisons between treatments yielded low intertreatment C(S) indices, indicating treatment-dependent alterations in banding patterns, whereas intratreatment comparisons revealed increased homogeneity in antibiotic-treated vs. control pigs. Sequence analysis of treatment-specific bands identified three Lactobacillus, one Streptococcus, and one Bacillus species that were diminished with antibiotic rotation treatment, whereas tylosin selected for the presence of L. gasseri. Lactobacillus-specific qPCR was performed and analyzed as a percentage of total bacteria to further evaluate the effects of antibiotic administration on this genus. Total bacteria were decreased (P < 0.05) by tylosin and rotation treatments, whereas the percentage of lactobacilli increased (P < 0.05) by d 14 and through d 28 in tylosin-treated pigs. The decrease in total bacteria by antibiotics may reduce host-related intestinal or immune responses, which would divert energy that could otherwise be used for growth. Conversely, the ability of tylosin to improve animal growth may relate to its apparent selection for lactobacilli, commensals known to competitively exclude potentially pathogenic species from colonizing the intestine.

Molecular ecological analysis of porcine ileal microbiota responses to antimicrobial growth promoters.

Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens. Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and oats. Outbreaks of NE can be prevented or treated by the use of in-feed antibiotics. However, the current debate regarding the prophylactic use of antibiotics in animal diets necessitates a better understanding of factors that influence intestinal colonization by C. perfringens as well as the pathophysiological consequences of its growth. We report a study with a chick model of NE, which used molecular (16S rRNA gene [16S rDNA]) and culture-based microbiological techniques to investigate the impact of the macrolide antibiotic tylosin phosphate (100 ppm) and a dietary NSP (pectin) on the community structure of the small intestinal microbiota relative to colonization by C. perfringens. The effects of tylosin and pectin on mucolytic activity of the microbiota and C. perfringens colonization and their relationship to pathological indices of NE were of particular interest. The data demonstrate that tylosin reduced the percentage of mucolytic bacteria in general and the concentration of C. perfringens in particular, and these responses correlated in a temporal fashion with a reduction in the occurrence of NE lesions and an improvement in barrier function. The presence of pectin did not significantly affect the variables measured. Thus, it appears that tylosin can control NE through its modulation of C. perfringens colonization and the mucolytic activity of the intestinal microbiota.

https://aac.asm.org/content/aac/47/10/3311.full.pdf

Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis

Supplementation of infant formulas with prebiotic ingredients continues the effort to mimic functional properties of human milk. In this double-blind, controlled, 28-day study, healthy term infants received control formula (control group; n = 25) or control formula supplemented with polydextrose (PDX) and galactooligosaccharide (GOS) (4 g/liter) (PG4 group; n = 27) or with PDX, GOS, and lactulose (LOS) (either 4 g/liter [PGL4 group; n = 27] or 8 g/liter [PGL8 group; n = 25]). A parallel breast-fed group (BF group) (n = 30) was included. Stool characteristics, formula tolerance, and adverse events were monitored. Fecal bacterial subpopulations were evaluated by culture-based selective enumeration (Enterobacteriaceae), quantitative real-time PCR (Clostridium clusters I, XI, and XIV, Lactobacillus, and Bifidobacterium), and fluorescence in situ hybridization (FISH) (Bifidobacterium). Fecal bacterial community profiles were examined by using 16S rRNA gene PCR-denaturing gradient gel electrophoresis. The daily stool consistency was significantly softer or looser in the BF group than in all of the groups that received formula. The formulas were well tolerated, and the incidences of adverse events did not differ among feeding groups. Few significant changes in bacterial subpopulations were observed at any time point. The bacterial communities were stable; individual profiles tended to cluster by subject rather than by group. Post hoc analysis, however, demonstrated that the bacterial community profiles for subjects in the BF, PG4, PGL4, and PGL8 groups that first received formula at a younger age were less stable than the profiles for subjects in the same groups that received formula at an older age, but there was no difference for the control group. These data indicate that formulas containing PDX, GOS, and LOS blends are more likely to influence gut microbes when administration is begun in early infancy and justify further investigation of the age-related effects of these blends on fecal microbiota.

https://aem.asm.org/content/aem/75/4/1121.full.pdf

Chad T. Collier

Papers

Antibiotics as growth promotants:mode of action

Molecular ecological analysis of the gastrointestinal microbiota: a review.

Molecular ecological analysis of porcine ileal microbiota responses to antimicrobial growth promoters.

Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis

Molecular ecological analysis of fecal bacterial populations from term infants fed formula supplemented with selected blends of prebiotics.