Evaluation of Broiler Litter with Reference to the Microbial Composition as Assessed by Using 16S rRNA and Functional Gene Markers

Abstract

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10 9 CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G؉C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.

Gram-positive bacteria are a major reservoir of Class 1 antibiotic resistance integrons in poultry litter

“…This explanation was ruled out by finding that 10 Gram-negative isolates from five distinct sampling times on both farms had only single copies of each of these genes (data not shown). Further, the possibility that cultivation had vastly underestimated the aerobic Gram-negative bacteria in litter was ruled out by a 16S rDNA library survey (9) that showed that ␥-proteobacteria, which includes Enterobacteriaceae, comprise Ͻ 2% of the litter community; none of the 340 clones examined in that study were Enterobacteriaceae, suggesting that they are a very minor component of the ␥-proteobacteria population in litter, just as they are in the bowel (41). In addition, litter concentration of the Q gene of the lysogenic coliphage, , in a random subset of samples from both farms differed by no more than 2-fold from that of tnsB, intI2, and MacConkey cfu (data not shown).…”

“…The resulting mixture, called poultry litter, is often replaced with fresh wood shavings between flocks and constitutes a significant component of waste from commercial poultry production (8). Litter has a unique largely aerobic microbiota, reflecting its various inocula and substrates (9). Rich in minerals, it can be recycled for fertilizer, among other uses (8).…”

The Potential of Antibiotic Alternatives for Enriching Beneficial Microbiota in Litter

“…The protocol used for isolating the community DNA from litter samples was as previously described [40]. PCR amplification of the V3 and V6 region of the bacterial 16S rRNA genes was conducted as previously described [42].…”

“…Selective media is often not as specific in some instances and too selective to allow certain bacteria to be detected. However the litter microbiota has been characterized by molecular ecology techniques in a few studies [17,40] and recently we reported the effects of probiotics or prebiotics on the microbiome and antibiotic resistome of broiler litter [42]. The objective of this report was to evaluate the impact of alternative growth promoters on the litter microbiota from a chick quality standpoint.…”

Development of a Polymerase Chain Reaction Assay for the Detection of Antibiotic Resistance Genes in Community DNA

“…All of these approaches, which rely on the culturing of specific bacteria, can be time consuming and expensive. More importantly, these isolation techniques are often highly selective, 17,23,29,39 and may miss most of the bacteria in a sample that are unculturable. 12,15,44 Studies using metagenomics and environmental samples to investigate unculturable bacteria also have been published.…”