scholarly journals Comparison of culture-based strategies for isolating bacteria from the porcine intestine

2021 ◽  
Author(s):  
Paul E. Moote ◽  
Danisa M. Bescucci ◽  
Rodrigo Ortega Polo ◽  
Richard R. E. Uwiera ◽  
G. Douglas Inglis
Keyword(s):  
Bacterial Species ◽  
Enteric Bacteria ◽  
Direct Plating ◽  
Uncultured Bacteria ◽  
Shannon’S Index ◽  
Term Enrichment ◽  
Model Animal ◽  
Novel Bacteria ◽  
Combination Of Methods

The isolation of bacteria that represent the diversity of autochthonous taxa in the gastrointestinal tract is necessary to fully ascertain function, but the majority of bacterial species inhabiting the intestines of mammals are fastidious, and thus challenging to isolate. The goal of the current study was to isolate a diverse assemblage of bacteria from the intestine of pigs as a model animal, and to comparatively examine various novel and traditional isolation strategies. Methods used included long-term enrichments, direct plating, a modified Ichip, as well as ethanol and Tyndallization treatment of samples to select for endospore-forming taxa. A total of 234 taxa (91 previously uncultured) comprising 80 genera and seven phyla were isolated from mucosal and luminal samples from the ileum, cecum, ascending colon, and spiral colon removed from animals under anesthesia. The diversity of bacteria isolated from the large intestine was less than detected by next-generation sequence analysis. Long-term enrichments yielded the greatest diversity of recovered bacteria (Shannon’s index [SI] = 4.7). Methods designed to isolate endospore-forming bacteria produced the lowest diversity (SI ≤ 2.7), with Tyndallization yielding a lower diversity than the ethanol method. However, the isolation frequency of previously uncultured bacteria was greatest for ethanol-treated samples (41.9%) and the Ichip method (32.5%). The goal of recovering a diverse collection of enteric bacteria was achieved. Importantly, study findings demonstrate that it is necessary to use a combination of methods in concert to isolate bacteria that are representative of the diversity within the intestines of mammals. IMPORTANCE This work determined that using a combination of isolation methods is necessary to increase the diversity of bacteria recovered from the intestines of monogastric mammals. Direct plating methods have traditionally been used to isolate enteric bacteria, and recent methods (e.g. diffusion methods [i.e. Ichip] or differential isolation of endospore-forming bacteria) have been suggested to be superior at increasing diversity, including the recovery of previously uncultured taxa. We showed that long-term enrichment of samples using a variety of media isolated the most diverse and novel bacteria. Application of the Ichip method delivered similar diversity of bacteria to enrichment and direct plating methods. Methods that selected for endospore-forming bacteria generated collections that differed in composition to other methods with reduced diversity. However, the ethanol treatment frequently isolated novel bacteria. By using a combination of methods in concert, a diverse collection of enteric bacteria was generated for ancillary experimentation.

scholarly journals Antimicrobial-Resistant Enteric Bacteria from Dairy Cattle

2006 ◽  
Vol 73 (1) ◽  
pp. 156-163 ◽  
Author(s):  
Ashish A. Sawant ◽  
Narasimha V. Hegde ◽  
Beth A. Straley ◽  
Sarah C. Donaldson ◽  
Brenda C. Love ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Dna Hybridization ◽  
Bacterial Species ◽  
Dairy Herd ◽  
Enteric Bacteria ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Predominant Species ◽  
Lactating Dairy Cattle

ABSTRACT A study was conducted to understand the descriptive and molecular epidemiology of antimicrobial-resistant gram-negative enteric bacteria in the feces of healthy lactating dairy cattle. Gram-negative enteric bacteria resistant to ampicillin, florfenicol, spectinomycin, and tetracycline were isolated from the feces of 35, 8, 5, and 42% of 213 lactating cattle on 74, 39, 9, 26, and 82% of 23 farms surveyed, respectively. Antimicrobial-resistant gram-negative bacteria accounted for 5 (florfenicol) to 14% (tetracycline) of total gram-negative enteric microflora. Nine bacterial species were isolated, of which Escherichia coli (87%) was the most predominant species. MICs showing reduced susceptibility to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline were observed in E. coli isolates. Isolates exhibited resistance to ampicillin (48%), ceftiofur (11%), chloramphenicol (20%), florfenicol (78%), spectinomycin (18%), and tetracycline (93%). Multidrug resistance (≥3 to 6 antimicrobials) was seen in 40% of E. coli isolates from healthy lactating cattle. Of 113 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 93% of isolates, while the remaining 7% isolates carried the tet(A) determinant. DNA-DNA hybridization assays revealed that tet determinants were located on the chromosome. Pulsed-field gel electrophoresis revealed that tetracycline-resistant E. coli isolates (n = 99 isolates) belonged to 60 subtypes, which is suggestive of a highly diverse population of tetracycline-resistant organisms. On most occasions, E. coli subtypes, although shared between cows within the herd, were confined mostly to a dairy herd. The findings of this study suggest that commensal enteric E. coli from healthy lactating cattle can be an important reservoir for tetracycline and perhaps other antimicrobial resistance determinants.

scholarly journals Antioxidative and antimicrobial potentials of Parmelia saxatilis and Pseudoevernia furfuracea

2016 ◽  
pp. 9-18
Author(s):  
Jasmina Cilerdzic ◽  
Mirjana Stajic ◽  
Jelena Vukojevic
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Side Effects ◽  
Klebsiella Pneumoniae ◽  
Bacterial Species ◽  
Phenol Content ◽  
Negative Side Effects ◽  
Highly Correlated ◽  
Ethanol Extracts

Even though numerous lichen species possess significant medical potentials they are still unexplored, and particularly species and strains originating from Serbia. Therefore, the aim of this study was to evaluate the antioxidative and antimicrobial potential of ethanol extracts of Parmelia saxatilis and Pseudoevernia furfuracea collected in Serbia. The tested extracts were good scavengers of DPPH radicals, with capacities ranging from 14.76% to 79.76% in P. saxatilis and from 21.39% to 90.04% in P. furfuracea. In P. saxatilis level of DPPH? neutralisation was highly correlated with phenol content (r2 = 0.9981) and in P. furfuracea with amount of total flavonoides (r2 = 0.9641). The extract of P. furfuracea inhibited the growth of all tested microorganisms with exception of Aspergillus flavus, while P. saxatilis extract affected only growth of bacterial species. Among tested microorganisms, Staphylococcus aureus and Klebsiella pneumoniae were the most sensitive, while Enterococcus faecalis, Pseudomonas aeruginosa as well as micromycetes were the least sensitive to tested extracts. Because of these potentials and the fact that their long term usage does not have any negative side effects on organism and development of microbial resistance, the extracts could be included in conventional therapy.

scholarly journals Diversity and evolution of surface polysaccharide synthesis loci in Enterobacteriales

2019 ◽  
Author(s):  
Kathryn E. Holt ◽  
Florent Lassalle ◽  
Kelly L. Wyres ◽  
Ryan Wick ◽  
Rafal J. Mostowy
Keyword(s):  
Evolutionary Dynamics ◽  
Bacterial Species ◽  
Purifying Selection ◽  
Evolutionary Forces ◽  
Polysaccharide Synthesis ◽  
Surface Polysaccharides ◽  
Surface Polysaccharide ◽  
Selective Preservation ◽  
Multiple Species

Bacterial capsules and lipopolysaccharides are diverse surface polysaccharides (SPs) that serve as the frontline for interactions with the outside world. While SPs can evolve rapidly, their diversity and evolutionary dynamics across different taxonomic scales has not been investigated in detail. Here, we focused on the bacterial order Enterobacteriales (including the medically-relevant Enterobacteriaceae), to carry out comparative genomics of two SP locus synthesis regions, cps and kps, using 27,334 genomes from 45 genera. We identified high-quality cps loci in 22 genera and kps in 11 genera, around 4% of which were detected in multiple species. We found SP loci to be highly dynamic genetic entities: their evolution was driven by high rates of horizontal gene transfer (HGT), both of whole loci and component genes, and relaxed purifying selection, yielding large repertoires of SP diversity. In spite of that, we found the presence of (near-)identical locus structures in distant taxonomic backgrounds that could not be explained by recent exchange, pointing to long-term selective preservation of locus structures in some populations. Our results reveal differences in evolutionary dynamics driving SP diversity within different bacterial species, with lineages of Escherichia coli, Enterobacter hormachei and Klebsiella aerogenes most likely to share SP loci via recent exchange; and lineages of Salmonella enterica, Citrobacter sakazakii and Serratia marcescens most likely to share SP loci via other mechanisms such as long-term preservation. Overall, the evolution of SP loci in Enterobacteriales is driven by a range of evolutionary forces and their dynamics and relative importance varies between different species.

scholarly journals Measurement and modelling of evaporation from a coastal wetland in Maputaland, South Africa

2012 ◽  
Vol 9 (2) ◽  
pp. 1741-1782 ◽  
Author(s):  
A. D. Clulow ◽  
C. S. Everson ◽  
M. G. Mengistu ◽  
C. Jarmain ◽  
G. P. W. Jewitt ◽  
...  
Keyword(s):  
Water Balance ◽  
Animal Species ◽  
Coastal Wetland ◽  
Surface Renewal ◽  
Site Specific ◽  
Combination Of Methods ◽  
A Site ◽  
Sr Method ◽  
Subtropical Environment

Abstract. The contribution of freshwater supply from the Mfabeni Mire to Lake St. Lucia during dry periods is important to the survival of certain plant and animal species in the iSimangaliso Wetland Park. This freshwater supply is mainly dependent on the variability of the major components of the water balance, namely rainfall and total evaporation (ET). Attempts to quantify the water balance have been limited through uncertainties in quantifying ET from the Mfabeni Mire. Despite advances in evaporation measurement and modelling from wetlands, there still exists some doubt as to which methods are best suited to characterise wetland ET with most authors suggesting a combination of methods. In this study, the surface renewal (SR) method was successfully used to determine the long-term ET (12 months) from the Mfabeni Mire with calibration using eddy covariance during two window periods of approximately one week each. The SR method was found to be inexpensive, reliable and with low power requirements for unattended operation. The annual ET was lower (900 mm yr−1) than expected, due to cloud cover in summer and low atmospheric demand throughout the year, despite the available water and high windspeeds. Daily ET estimates were compared to the Priestley-Taylor results and a site specific calibration α = 1.0 was obtained for the site. The Priestley-Taylor results agreed well with the actual ET from the surface renewal technique (R2 = 0.96) throughout the 12 month period. A monthly crop factor (Kc) was determined for the standardised FAO-56 Penman-Monteith. However, Kc was variable in some months and should be used with caution for daily ET modelling. These results represent not only some of the first long-term measurements of ET from a wetland in Southern Africa, but also one of the few studies of actual ET in a subtropical peatland in the Southern Hemisphere. The study provides wetland ecologists and hydrologists with guidelines for the use of two internationally applied models for the estimation of wetland ET within a coastal, subtropical environment.

scholarly journals Colonization of gut microbiota by plasmid-carrying bacteria is facilitated by evolutionary adaptation to antibiotic treatment

2021 ◽  
Author(s):  
Peng Zhang ◽  
Daqing Mao ◽  
Huihui Gao ◽  
Liyang Zheng ◽  
Zeyou Chen ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Bacterial Species ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Evolutionary Adaptation ◽  
Antibiotic Exposure ◽  
Resistance Protein ◽  
K 12

AbstractMultidrug-resistant plasmid-carrying bacteria are of particular clinical concern as they could transfer antibiotic resistance genes to other bacterial species. However, little is known whether evolutionary adaptation of plasmid-carrying bacteria after long-term antibiotic exposure could affect their subsequent colonization of the human gut. Herein, we combined a long-term evolutionary model based on Escherichia coli K-12 MG1655 and the multidrug-resistant plasmid RP4 with in vivo colonization experiments in mice. We found that the evolutionary adaptation of plasmid-carrying bacteria to antibiotic exposure facilitated colonization of the murine gut and subsequent plasmid transfer to gut bacteria. The evolved plasmid-carrying bacteria exhibited phenotypic alterations, including multidrug resistance, enhanced bacterial growth and biofilm formation capability and decreased plasmid fitness cost, which might be jointly caused by chromosomal mutations (SNPs in rpoC, proQ, and hcaT) and transcriptional modifications. The upregulated transcriptional genes, e.g., type 1 fimbrial-protein pilus (fimA and fimH) and the surface adhesin gene (flu) were likely responsible for the enhanced biofilm-forming capacity. The gene tnaA that encodes a tryptophanase-catalyzing indole formation was transcriptionally upregulated, and increased indole products participated in facilitating the maximum population density of the evolved strains. Furthermore, several chromosomal genes encoding efflux pumps (acriflavine resistance proteins A and B (acrA, acrB), outer-membrane protein (tolC), multidrug-resistance protein (mdtM), and macrolide export proteins A and B (macA, macB)) were transcriptionally upregulated, while most plasmid-harboring genes (conjugal transfer protein (traF) and (trbB), replication protein gene (trfA), beta-lactamase TEM precursor (blaTEM), aminoglycoside 3'-phosphotransferase (aphA) and tetracycline resistance protein A (tetA)) were downregulated. Collectively, these findings demonstrated that evolutionary adaptation of plasmid-carrying bacteria in an antibiotic-influenced environment facilitated colonization of the murine gut by the bacteria and plasmids.

scholarly journals Characterization of human norovirus binding to gut-associated bacterial ligands

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Erin A. Almand ◽  
Matthew D. Moore ◽  
Lee-Ann Jaykus
Keyword(s):  
Western Blot ◽  
Lectin Binding ◽  
Bacterial Species ◽  
Enteric Bacteria ◽  
Virus Binding ◽  
Binding Interaction ◽  
Human Norovirus ◽  
Ligand Identification ◽  
Strain Dependent

Abstract Objective Research suggests human norovirus binding to histo-blood group antigen (HBGA)-like molecules on enteric bacteria may enhance viral pathogenesis; however, the properties of these bacterial ligands are not well known. Previous work identified, but did not characterize, seven norovirus-binding bacteria. To further examine this bacteria–virus binding interaction, enteric bacteria were analyzed via Western blot with anti-HBGA antibodies and lectins targeting HBGA-associated sugar components. Virus overlay assays using capsids from six different human norovirus strains further identified responsible ligands and strain dependent binding properties. Results Each bacterial species possessed varying degrees of HBGA-like activity, and lectin binding further elucidated potential sugar residues involved (N-acetyl-galactosamine, α-d-galactose or α-l-fucose). Both GI and GII norovirus capsids bound specific bacterial ligand sizes, and generally corresponded to anti-HBGA Western blot patterns. A 35-kDa band reacted with all HBGA antibodies, bound all six of the noroviruses tested, and had a high affinity for the lectins. Collectively, this work characterizes the varying carbohydrate residues potentially responsible for norovirus–bacteria interactions and provides a basis for future ligand identification.

scholarly journals Group of peptides that act synergistically with hydrophobic antibiotics against gram-negative enteric bacteria.

1996 ◽  
Vol 40 (8) ◽  
pp. 1801-1805 ◽  
Author(s):  
M Vaara ◽  
M Porro
Keyword(s):  
Synthetic Peptide ◽  
Bacterial Species ◽  
Cetylpyridinium Chloride ◽  
Enteric Bacteria ◽  
Polymyxin B ◽  
Disulfide Bridge ◽  
Gram Negative ◽  
E Coli ◽  
Cationic Detergents ◽  
Lysine Residues

A synthetic peptide, KFFKFFKFFK [corrected], consisting of cationic lysine residues and hydrophobic phenylalanine residues was found to sensitize gram-negative bacteria to hydrophobic and amphipathic antibiotics. At a concentration of 3 micrograms/ml, it decreased the MIC of rifampin for smooth, encapsulated Escherichia coli by a factor of 300. Other susceptible bacterial species included Enterobacter cloacae, Klebsiella pneumoniae, and Salmonella typhimurium, but Pseudomonas aeruginosa was resistant. Similar results were obtained with another synthetic peptide, IKFLKFLKFLK [corrected]. The fractional inhibitory concentration indices for the synergism of these peptides with rifampin, erythromycin, fusidic acid, and novobiocin were very close to those determined for the previously characterized potent outer-membrane-disorganizing agents polymyxin B nonapeptide and deacylpolymyxin B. KFFKFFKFFK [corrected] had direct activity against the gram-positive organism Micrococcus strain ML36, was strongly hemolytic, and was as active on polymyxin-resistant E. coli mutants as on their parent. These three attributes made KFFKFFKFFK [corrected] different from polymyxin derivatives and similar to cationic detergents, such as cetylpyridinium chloride. However, whereas the MIC of cetylpyridinium chloride for E. coli is low (0.5 to 4 micrograms/ml), that of KFFKFFKFFK [corrected] is much higher (30 to 100 micrograms/ml). Other groups of synthetic peptides studied included polymyxin-like peptides with an intrachain disulfide bridge. Their synergism with antibiotics was less marked. Still other peptides, including KEKEKEKEKE and KKKKKKFLFL, lacked any synergism with the probe antibiotics.

Thermophilic two-phase anaerobic digestion of source-sorted organic fraction of municipal solid waste for bio-hythane production: effect of recirculation sludge on process stability and microbiology over a long-term pilot-scale experience

2014 ◽  
Vol 69 (11) ◽  
pp. 2200-2209 ◽  
Author(s):  
A. Giuliano ◽  
L. Zanetti ◽  
F. Micolucci ◽  
C. Cavinato
Keyword(s):  
Anaerobic Digestion ◽  
Municipal Solid Waste ◽  
Solid Waste ◽  
Bacterial Species ◽  
Pilot Scale ◽  
Dark Fermentation ◽  
Organic Fraction ◽  
Recirculation Flow ◽  
Production Of Hydrogen

A two-stage thermophilic anaerobic digestion process for the concurrent production of hydrogen and methane through the treatment of the source-sorted organic fraction of municipal solid waste was carried out over a long-term pilot scale experience. Two continuously stirred tank reactors were operated for about 1 year. The results showed that stable production of bio-hythane without inoculum treatment could be obtained. The pH of the dark fermentation reactor was maintained in the optimal range for hydrogen-producing bacteria activity through sludge recirculation from a methanogenic reactor. An average specific bio-hythane production of 0.65 m3 per kg of volatile solids fed was achieved when the recirculation flow was controlled through an evaporation unit in order to avoid inhibition problems for both microbial communities. Microbial analysis indicated that dominant bacterial species in the dark fermentation reactor are related to the Lactobacillus family, while the population of the methanogenic reactor was mainly composed of Defluviitoga tunisiensis. The archaeal community of the methanogenic reactor shifted, moving from Methanothermobacter-like to Methanobacteriales and Methanosarcinales, the latter found also in the dark fermentation reactor when a considerable methane production was detected.

scholarly journals Single-colony sequencing reveals microbe-by-microbiome phylosymbiosis between the cyanobacterium Microcystis and its associated bacteria

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Olga M. Pérez-Carrascal ◽  
Nicolas Tromas ◽  
Yves Terrat ◽  
Elisa Moreno ◽  
Alessandra Giani ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Heterotrophic Bacteria ◽  
Bacterial Species ◽  
Colony Growth ◽  
Genomic Diversity ◽  
Gene Copy ◽  
Metagenomic Sequencing ◽  
Associated Bacteria ◽  
Microbiome Composition

Abstract Background Cyanobacteria from the genus Microcystis can form large mucilaginous colonies with attached heterotrophic bacteria—their microbiome. However, the nature of the relationship between Microcystis and its microbiome remains unclear. Is it a long-term, evolutionarily stable association? Which partners benefit? Here we report the genomic diversity of 109 individual Microcystis colonies—including cyanobacteria and associated bacterial genomes—isolated in situ and without culture from Lake Champlain, Canada and Pampulha Reservoir, Brazil. Results We identified 14 distinct Microcystis genotypes from Canada, of which only two have been previously reported, and four genotypes specific to Brazil. Microcystis genetic diversity was much greater between than within colonies, consistent with colony growth by clonal expansion rather than aggregation of Microcystis cells. We also identified 72 bacterial species in the microbiome. Each Microcystis genotype had a distinct microbiome composition, and more closely related genotypes had more similar microbiomes. This pattern of phylosymbiosis could be explained by co-phylogeny in only two out of the nine most prevalent associated bacterial genera, Roseomonas and Rhodobacter. These phylogenetically associated genera could enrich the metabolic repertoire of Microcystis, for example by encoding the biosynthesis of complementary carotenoid molecules. In contrast, other colony-associated bacteria showed weaker signals of co-phylogeny, but stronger evidence of horizontal gene transfer with Microcystis. These observations suggest that acquired genes are more likely to be retained in both partners (Microcystis and members of its microbiome) when they are loosely associated, whereas one gene copy is sufficient when the association is physically tight and evolutionarily long-lasting. Conclusions We have introduced a method for culture-free isolation of single colonies from nature followed by metagenomic sequencing, which could be applied to other types of microbes. Together, our results expand the known genetic diversity of both Microcystis and its microbiome in natural settings, and support their long-term, specific, and potentially beneficial associations.

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