Swine-manure Storage Pit Research Articles

Comparison of antibiotic resistance genes in swine manure storage pits of Iowa, USA

Antimicrobial resistance (AMR) can develop in deep-pit swine manure storage when bacteria are selectively pressured by unmetabolized antibiotics. Subsequent manure application on row crops is then a source of AMR into soil and downstream runoff water. Therefore, understanding the patterns of diverse antibiotic resistance genes (ARGs) in manure among different farms is important for both interpreting the results of the detection of these genes from previous studies and for the use of these genes as bioindicators of manure borne antibiotic resistance in the environment. Previous studies of manure-associated ARGs are based on limited samples of manures. To better understand the distribution of ARGs between manures, we characterized manures from 48 geographically independent swine farms across Iowa. The objectives of this study were to characterize the distribution of ARGs among these manures and to evaluate what factors in manure management may influence the presence of ARGs in manures. Our analysis included quantification of two commonly found ARGs in swine manure, ermB and tetM. Additionally, we characterized a broader suite of 31 ARGs which allowed for simultaneous assays of the presence or absence of multiple genes. We found the company integrator had a significant effect on both ermB (P=0.0007) and tetM gene concentrations (P=0.0425). Our broad analysis on ARG profiles found that the tet(36) gene was broadly present in swine manures, followed by the detection of tetT, tetM, erm(35), ermF, ermB, str, aadD, and intl3 in samples from 14 farms. Finally, we provide a comparison of methods to detect ARGs in manures, specifically comparing conventional and high-throughput qPCR and discuss their role in ARG environmental monitoring efforts. Results of this study provide insight into commonalities of ARG presence in manure holding pits and provide supporting evidence that company integrator decisions may impact ARG concentrations.

Genome Sequence of a Novel Multiple-Antibiotic-Resistant Member of the Erysipelotrichaceae Family Isolated from a Swine Manure Storage Pit

The swine gastrointestinal tract and stored swine manure may serve as reservoirs of antibiotic resistance genes, as well as sources of novel bacteria. Here, we report the draft genome sequence of a novel taxon in the Erysipelotrichaceae family, isolated from a swine manure storage pit that is resistant to multiple antibiotics.

Robinsoniella peoriensis, originally isolated from swine manure, and early periprosthetic hip infection: Case report and review of the literature

We report on the first case of a periprosthetic joint infection with the anaerobic spore-forming Gram-positive rod Robinsoniella peoriensis as the causative agent. The bacterium was first isolated from a swine manure storage pit and has so far rarely been associated with human infections.

Savagea faecisuis gen. nov., sp. nov., a tylosin- and tetracycline-resistant bacterium isolated from a swine-manure storage pit.

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular methods was performed on three strains of a Gram-stain positive, non-sporeforming, motile aerobic rod-shaped bacterium resistant to tylosin and tetracycline isolated from a swine-manure storage pit. On the basis of 16S rRNA gene sequence analyses, it was confirmed that these isolates are highly related to each other and form a hitherto unknown lineage within the Planococcaceae. In particular, pairwise analysis of the 16S rRNA gene sequence demonstrated that the novel organism is closely related to members of the genus Sporosarcina (92.8-94.5 %), Pyschrobacillus (93.5-93.9 %) and Paenisporosarcina (93.3-94.5 %). The predominant fatty acids were found to consist of iso-C15:0 and iso-C17:1 ω10c and the G+C mol% was determined to be 41.8. Based on biochemical, chemotaxonomic, and phylogenetic evidence, it is proposed that these novel strains be classified as a novel genus and species, Savagea faecisuis gen nov., sp. nov. The type strain is Con12(T) (=CCUG 63563(T) = NRRL B-59945(T) = NBRC 109956(T)).

Erratum to: Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular genetic methods was performed on six strains of an unknown Gram-positive, nonspore-forming, facultative anaerobic coccus-shaped bacterium isolated from a swine-manure storage pit. On the basis of 16S rRNA, RNA polymerase-subunit (rpoA), and the 60-kilodalton chaperonin (cpn60) gene sequence analyses, it was shown that all the isolates were enterococci but formed two separate lines of descent. Pairwise 16S rRNA sequence comparisons demonstrated that the two novel organisms were most closely related to each other (97.9%) and to Enterococcus aquimarinus (97.8%). Both organisms contained major amounts of C16:0, C16:1 ω7c, and C18:1 ω7c/12t/9t as the major cellular fatty acids. Based on biochemical, chemotaxonomic, and phylogenetic evidence, the names Enterococcus lemanii sp. nov. (type strain PC32(T)=CCUG 61260(T)=NRRL B-59661(T)) and Enterococcus eurekensis sp. nov. (type strain PC4B(T)=CCUG 61259(T)=NRRL B-59662(T)) are proposed for the hitherto undescribed species.

Erratum to: Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit

Erratum to: Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit

First Korean Case of Robinsoniella peoriensis Bacteremia in a Patient with Aspiration Pneumonia

Robinsoniella peoriensis has recently been identified as a Gram-positive, spore-forming, anaerobic rod originally recovered from swine manure storage pits. To date, 6 cases of R. peoriensis infection have been reported, including 2 cases of bacteremia, 1 of abdominal fluid collection, and 3 of wound infection. In the present study, we report a 76-yr-old man with R. peoriensis bacteremia who developed aspiration pneumonia. Gram staining of a purified colony revealed Gram-positive, rod-shaped bacteria. Biochemical identification using API 20 A (bioMérieux, France) indicated presence of Clostridium spp. We performed both 500-bp and full-gene sequencing of 16S rRNA of the isolate. The sequence was analyzed with MicroSeq ID 16S rRNA Library v2.0 (Applied Biosystems, USA), GenBank Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/genbank), and EzTaxon database v2.1 (http://www.eztaxon.org). The 500-bp 16S rRNA sequence of the blood culture isolate showed 99.16-99.79% similarity with R. peoriensis and the full-gene 16S rRNA sequence showed 98.87-99.50% similarity with R. peoriensis. The organism was confirmed as R. peoriensis by using all of the mentioned databases except for MicroSeq, which did not include the RNA sequence of this bacterium. This case suggests that identification of R. peoriensis might be challenging in clinical laboratories with no access to molecular methods, as certain commercial identification systems may not identify, or may misidentify, this organism. To the best of our knowledge, this is the first report of the isolation of R. peoriensis in Korea.

Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular genetic methods was performed on six strains of unknown Gram-positive, nonspore-forming, facultative anaerobic coccus-shaped bacteria isolated from a swine-manure storage pit. On the basis of the 16S rRNA, RNA polymerase α-subunit (rpoA) and 60 kDa chaperonin (cpn60) gene sequence analyses, it was shown that all the isolates were enterococci but formed two separate lines of descent. Pairwise 16S rRNA gene sequence comparisons demonstrated that the two novel organisms were most closely related to each other (97.9 %) and to Enterococcus aquimarinus (97.8 %). Both organisms contained major amounts of C(16:0), C(16:1) ω7c, C(16:1) ω7c, and C(18:1) ω7c/12t/9t as the major cellular fatty acids. Based on biochemical, chemotaxonomic and phylogenetic evidence, the names Enterococcus lemanii sp. nov. (type strain PC32(T) = CCUG 61260(T) = NRRL B-59661(T)) PPC27A = CCUG 61369; PPC38 = CCUG 61261 [corrected] and Enterococcus eurekensis sp. nov. (type strain PC4B(T) = CCUG 61259(T) = NRRL B-59662(T)) PPC15 = CCUG 61368; PPC107 = CCUG 61372 [corrected] are proposed for these hitherto undescribed species.

Robinsoniella peoriensis infection following surgery for scoliosis: a case report

IntroductionRobinsoniella peoriensis was recently identified as a Gram-positive, spore-forming, anaerobic bacillus originally isolated from swine manure storage pits. Seven isolates have been subsequently reported from human sources.Case presentationWe report the case of an infection caused by R. peoriensis in a 45-year-old Caucasian woman after posterior instrumentation correction of idiopathic thoracolumbar scoliosis. The identification was made by culture of samples inoculated onto blood agar and chocolate agar and was confirmed by 16 S ribosomal ribonucleic acid gene sequencing.ConclusionsWe discuss similar cases suggesting that R. peoriensis is responsible for health care-associated infections with the colonic flora as a potential source of infection.

Peptostreptococcus russellii sp. nov., isolated from a swine-manure storage pit

Using a polyphasic approach, a taxonomic study was performed on seven strains of an unknown Gram-reaction-positive, non-spore-forming, obligately anaerobic coccus-shaped bacterium, isolated from a swine-manure storage pit. Comparative 16S rRNA gene sequencing confirmed that all seven isolates were highly related to each other and formed a hitherto unknown lineage within the clostridial rRNA XI cluster of organisms. Pairwise analysis demonstrated that the novel organism was most closely related to Peptostreptococcus anaerobius CCUG 7835(T) and Peptostreptococcus stomatis CCUG 51858(T) with 16S rRNA gene sequence similarities of 95.5 and 93.0 %, respectively. The peptidoglycan type of the cell wall was determined to be A4α l-Lys-d-Asp and glucose, xylose and traces of mannose were detected as the cell-wall sugars. Based on biochemical, chemotaxonomic and phylogenetic evidence the unknown bacterium represents a new species of the genus Peptostreptococcus, for which the name Peptostreptococcus russellii sp. nov, is proposed. The type strain is RT-10B(T) ( = CCUG 58235(T) = NRRL B-59380(T) = DSM 23041(T)).

Robinsoniella peoriensis Bacteremia in a Patient with Pancreatic Cancer

Robinsoniella peoriensis was recently identified as a Gram-positive, spore-forming, anaerobic rod originally isolated from swine manure storage pits. We describe here a case of R. peoriensis bacteremia in a 42-year-old male with pancreatic cancer. The identification was confirmed by unique phenotypic profiles and partial 16S rRNA gene sequences.

Robinsoniella peoriensis gen. nov., sp. nov., isolated from a swine-manure storage pit and a human clinical source

A polyphasic taxonomic study was performed on six strains of an unknown Gram-positive, non-motile, spore-forming, short oval to rod-shaped bacterium isolated from a swine-manure storage pit. In addition to these strains, an isolate deposited in the Culture Collection of the University of Göteborg (Sweden) was found to be biochemically related to the manure strains. The major end products of metabolism included acetate and succinate but not butyrate. Comparative 16S rRNA gene sequencing confirmed that all these isolates were closely related to each other and formed a hitherto unknown lineage within the clostridial rRNA XIVa cluster of organisms. On the basis of phylogenetic, biochemical and phenotypic evidence, it is proposed that the unknown bacterium represents a novel genus and species, for which the name Robinsoniella peoriensis gen. nov., sp. nov. is proposed. The type strain of Robinsoniella peoriensis is PPC31T (=CCUG 48729T =NRRL B-23985T).

Bacteroides propionicifaciens sp. nov., isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms

Two strictly anaerobic bacterial strains (SV434(T) and S562) were isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms in Japan. They had identical 16S rRNA gene sequences and showed almost the same phenotypic properties. The cells of both strains were Gram-negative, non-motile, non-spore-forming rods; extraordinarily long rods often occurred. Remarkable stimulation of growth occurred with the addition of haemin and cobalamin (vitamin B(12)) to the medium. The supplementary cobalamin and haemin could be replaced if autoclaved and clarified sludge fluid obtained from the reactor was added. Both strains utilized a range of growth substrates, including arabinose, fructose, galactose, glucose, mannose, cellobiose, maltose, glycogen, starch, dextrin, amygdalin, lactate and pyruvate. Both strains produced acetate and propionate with a small amount of succinate from these substrates in the presence of haemin and cobalamin. Both strains were slightly alkaliphilic, having a pH optimum at about 7.9. The temperature range for growth was 5-35 degrees C, the optimum being 30 degrees C. The NaCl concentration range for growth was 0-4 % (w/v). Catalase activity was not detected in cells cultivated without haemin, whereas cells cultivated with haemin usually had the enzyme activity. Oxidase and nitrate-reducing activities were not detected. Aesculin was hydrolysed, but gelatin was not hydrolysed. Both strains were sensitive to bile acids. The major cellular fatty acids of both strains were anteiso-C(15 : 0) and iso-C(15 : 0). Menaquinones MK-8(H(0)) and MK-9(H(0)) were the major respiratory quinones and the genomic DNA G+C contents were 46.2-47.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequences placed both strains in the phylum Bacteroidetes. Bacteroides coprosuis (isolated from swine-manure storage pits) was the species most closely related to both strains (95.9 % 16S rRNA gene sequence similarity to the type strain). On the basis of the phylogenetic, physiological and chemotaxonomic analyses, strains SV434(T) and S562 represent a novel species of the genus Bacteroides, for which the name Bacteroides propionicifaciens sp. nov. is proposed. The type strain is SV434(T) (=JCM 14649(T) =DSM 19291(T)).

Vagococcus elongatus sp. nov., isolated from a swine-manure storage pit

A polyphasic taxonomic study was performed on an uncharacterized Gram-positive, catalase-negative, elongated coccus-shaped bacterium isolated from a swine-manure storage pit. The bacterium, designated strain PPC9(T), was facultatively anaerobic and had a DNA G+C content of 44.5 mol%. Comparative 16S rRNA gene sequencing indicated that the bacterium represented a novel subline within the genus Vagococcus, close to but distinct from Vagococcus lutrae. Strain PPC9(T) was readily distinguished from the five recognized species of the genus Vagococcus by using biochemical tests and molecular genetic analysis. Based on phylogenetic and phenotypic evidence strain PPC9(T) is considered to represent a novel species of the genus Vagococcus, for which the name Vagococcus elongatus sp. nov. is proposed. The type strain is PPC9(T) (=CCUG 51432(T)=NRRL B-41357(T)).

Bacteroides coprosuis sp. nov., isolated from swine-manure storage pits

Two Gram-negative, anaerobic, non-spore-forming, rod-shaped organisms were isolated from a swine-manure storage pit. Based on morphological and biochemical criteria, the strains were tentatively identified as belonging to the genus Bacteroides but they did not appear to correspond to any recognized species of the genus. Comparative 16S rRNA gene sequencing studies showed that the strains were related closely to each other and confirmed their placement in the genus Bacteroides, but sequence divergence values of >10 % from reference Bacteroides species demonstrated that the organisms from manure represent a novel species. Based on biochemical criteria and molecular genetic evidence, it is proposed that the unknown isolates from manure be assigned to a novel species of the genus Bacteroides, as Bacteroides coprosuis sp. nov. The type strain is PC139T (=CCUG 50528T=NRRL B-41113T).

Application of group specific amplified rDNA restriction analysis to characterize swine fecal and manure storage pit samples

Group specific amplified ribosomal-DNA restriction analysis was evaluated as a method to rapidly assess microbial community structure in swine fecal and manure storage pit samples. PCR primer sequences were evaluated for their specificity to ribosomal DNA from selected bacterial groups by optimizing annealing temperatures and determining specificity using a set of primer target and non-target organisms. A number of primer sets were identified targeting the following groups: Bacteroides–Prevotella, clostridial clusters I and II, clostridial clusters IX and XI, clostridial clusters XIVa and XIVb, Lactobacillus, Desulfovibrionaceae and Streptococcus–Lactococcus, as well as an universal primer set to represent total populations. Each bacterial group was digested with at least three restriction enzymes. We applied the group specific amplified ribosomal-DNA restriction analysis to swine fecal and manure storage pit samples obtained on two separate occasions. Fecal and manure storage pit samples obtained on the same day were more similar to each other than to any other samples. Results were consistent with 16S ribosomal DNA sequencing data from bacterial isolates and clones obtained from swine feces and manure storage pit. The group specific amplified ribosomal-DNA restriction analysis technique was able to rapid detect gross bacterial community differences among swine fecal and manure storage pit samples and determine groups of interest for more detailed examination.

Isolation and identification of hyper-ammonia producing bacteria from swine manure storage pits.

Storage of swine manure is associated with the microbiological production of a variety of odorous compounds, including ammonia, organic acids, and alcohols, phenolics, and sulfides. Until recently, little was known about the microorganisms responsible for their production. Results from our laboratory have demonstrated that the predominant microbial populations of stored swine manure are anaerobic, low (G + C), Gram-positive bacteria. However, studies on pure cultures isolated from manure have found few microorganisms that produce appreciable ammonia concentrations. Therefore, selective and enrichment techniques were employed to isolate ammonia-producing bacteria from stored swine manure by using media containing peptone and amino acids as carbon and energy sources. We now report on the isolation of 40 bacterial cultures, a number of which are capable of producing at least 40 mM ammonia in peptone-amino acid medium, concentrations similar to those produced by hyper-ammonia producing (HAP) bacteria isolated from the rumen of cattle. The manure HAP isolates are phylogenetically distinct from the ruminal isolates and may prove to be intimately involved in the production of ammonia during storage of swine manure.

Identification of a new ribosomal protection type of tetracycline resistance gene, tet(36), from swine manure pits.

Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.