Assessing Genetic Heterogeneity within Bacterial Species Isolated from Gastrointestinal and Environmental Samples: How Many Isolates Does It Take?

ABSTRACT Strain typing of bacterial isolates is increasingly used to identify sources of infection or product contamination and to elucidate routes of transmission of pathogens or spoilage organisms. Usually, the number of bacterial isolates belonging to the same species that is analyzed per sample is determined by convention, convenience, laboratory capacity, or financial resources. Statistical considerations and knowledge of the heterogeneity of bacterial populations in various sources can be used to determine the number of isolates per sample that is actually needed to address specific research questions. We present data for intestinal Escherichia coli, Listeria monocytogenes, Klebsiella pneumoniae, and Streptococcus uberis from gastrointestinal, fecal, or soil samples characterized by ribotyping, pulsed-field gel electrophoresis, and PCR-based strain-typing methods. In contrast to previous studies, all calculations were performed with a single computer program, employing software that is freely available and with in-depth explanation of the choice and derivation of prior distributions. Also, some of the model assumptions were relaxed to allow analysis of the special case of two (groups of) strains that are observed with different probabilities. Sample size calculations, with a Bayesian method of inference, show that from 2 to 20 isolates per sample need to be characterized to detect all strains that are present in a sample with 95% certainty. Such high numbers of isolates per sample are rarely typed in real life due to financial or logistic constraints. This implies that investigators are not gaining maximal information on strain heterogeneity and that sources and transmission pathways may go undetected.

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Bacterial population genetics, evolution and epidemiology

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1999.0423 ◽

1999 ◽

Vol 354 (1384) ◽

pp. 701-710 ◽

Cited By ~ 123

Author(s):

Brian G. Spratt ◽

Martin C. J. Maiden

Keyword(s):

Population Structure ◽

De Novo ◽

Bacterial Species ◽

Epidemiological Studies ◽

Bacterial Isolates ◽

Bacterial Populations ◽

Relative Contribution ◽

Population Structures

Asexual bacterial populations inevitably consist of an assemblage of distinct clonal lineages. However, bacterial populations are not entirely asexual since recombinational exchanges occur, mobilizing small genome segments among lineages and species. The relative contribution of recombination, as opposed to de novo mutation, in the generation of new bacterial genotypes varies among bacterial populations and, as this contribution increases, the clonality of a given population decreases. In consequence, a spectrum of possible population structures exists, with few bacterial species occupying the extremes of highly clonal and completely non–clonal, most containing both clonal and non–clonal elements. The analysis of collections of bacterial isolates, which accurately represent the natural population, by nucleotide sequence determination of multiple housekeeping loci provides data that can be used both to investigate the population structure of bacterial pathogens and for the molecular characterization of bacterial isolates. Understanding the population structure of a given pathogen is important since it impacts on the questions that can be addressed by, and the methods and samples required for, effective molecular epidemiological studies.

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DIVERSITY OF CULTURABLE BACTERIAL MICROBIOTA OF THE Eisenia foetida DIGESTIVE TRACT

Revista Fitotecnia Mexicana ◽

10.35196/rfm.2018.3.255-264 ◽

2018 ◽

Vol 41 (3) ◽

pp. 255-264 ◽

Cited By ~ 1

Author(s):

J. Abraham Pérez-Pérez ◽

David Espinosa-Victoria ◽

Hilda V. Silva-Rojas ◽

Lucía López-Reyes

Keyword(s):

Bacterial Diversity ◽

Digestive Tract ◽

Bacterial Species ◽

Brain Heart Infusion ◽

Rrna Gene ◽

Eisenia Foetida ◽

Bacterial Isolates ◽

Bacterial Microbiota ◽

Decomposition Of Organic Matter ◽

Earthworm Gut

Bacteria are an unavoidable component of the natural earthworm diet; thus, bacterial diversity in the earthworm gut is directly linked to decomposition of organic matter and development of the surrounding plants. The aim of this research was to isolate and to identify biochemically and molecularly the culturable bacterial microbiota of the digestive tract of Eisenia foetida. Earthworms were sourced from Instituto de Reconversión Productiva y Bioenergética (IRBIO) and Colegio de Postgraduados (COLPOS), México. Bacterial isolation was carried out on plates of Brain Heart Infusion (BHI) culture medium. Fifty six and 44 bacterial isolates were obtained from IRBIO and COLPOS, respectively. The population was composed of 44 Gram-negative and 56 Gram-positive isolates. Over 50 % of the bacterial isolates were rod-shaped cells. The 16S rRNA gene was sequenced and nine genera were identified in worms from IRBIO (Bacillus, Paenibacillus, Solibacillus, Staphylococcus, Arthrobacter, Pantoea, Stenotrophomonas, Acinetobacter and Aeromonas) and six in worms from COLPOS (Bacillus, Paenibacillus, Stenotrophomonas, Staphylococcus, Acinetobacter and Aeromonas). Bacillus was the predominant genus, with eight and six species in the oligochaetes from IRBIO and COLPOS, respectively. The most represented bacteria in the worms from both sites were Bacillus sp. and B. subtilis. The predominance of Bacillus was probably due to spore formation, a reproductive strategy that ensures survival and dispersion in the soil and oligochaetes digestive tract. The gut of E. foetida not only harbored bacterial species of agronomic importance but also species potentially pathogenic for humans (Staphylococcus warneri, Pantoea agglomerans and Stentrophomonas sp.). The larger bacterial diversity in worms from IRBIO could be due to their feeding on cattle manure, which is a rich source of bacteria.

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Black Queen Evolution and Trophic Interactions Determine Plasmid Survival after the Disruption of the Conjugation Network

mSystems ◽

10.1128/msystems.00104-18 ◽

2018 ◽

Vol 3 (5) ◽

Cited By ~ 6

Author(s):

Johannes Cairns ◽

Katariina Koskinen ◽

Reetta Penttinen ◽

Tommi Patinen ◽

Anna Hartikainen ◽

...

Keyword(s):

Antibiotic Resistance ◽

Bacterial Communities ◽

Resistance Mechanism ◽

Real Life ◽

Ecological Factors ◽

Mobile Genetic Elements ◽

Resistance Mechanisms ◽

Antibiotics Resistance ◽

Bacterial Populations ◽

Genetic Elements

ABSTRACTMobile genetic elements such as conjugative plasmids are responsible for antibiotic resistance phenotypes in many bacterial pathogens. The ability to conjugate, the presence of antibiotics, and ecological interactions all have a notable role in the persistence of plasmids in bacterial populations. Here, we set out to investigate the contribution of these factors when the conjugation network was disturbed by a plasmid-dependent bacteriophage. Phage alone effectively caused the population to lose plasmids, thus rendering them susceptible to antibiotics. Leakiness of the antibiotic resistance mechanism allowing Black Queen evolution (i.e. a “race to the bottom”) was a more significant factor than the antibiotic concentration (lethal vs sublethal) in determining plasmid prevalence. Interestingly, plasmid loss was also prevented by protozoan predation. These results show that outcomes of attempts to resensitize bacterial communities by disrupting the conjugation network are highly dependent on ecological factors and resistance mechanisms.IMPORTANCEBacterial antibiotic resistance is often a part of mobile genetic elements that move from one bacterium to another. By interfering with the horizontal movement and the maintenance of these elements, it is possible to remove the resistance from the population. Here, we show that a so-called plasmid-dependent bacteriophage causes the initially resistant bacterial population to become susceptible to antibiotics. However, this effect is efficiently countered when the system also contains a predator that feeds on bacteria. Moreover, when the environment contains antibiotics, the survival of resistance is dependent on the resistance mechanism. When bacteria can help their contemporaries to degrade antibiotics, resistance is maintained by only a fraction of the community. On the other hand, when bacteria cannot help others, then all bacteria remain resistant. The concentration of the antibiotic played a less notable role than the antibiotic used. This report shows that the survival of antibiotic resistance in bacterial communities represents a complex process where many factors present in real-life systems define whether or not resistance is actually lost.

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Ice nucleation activity of bacteria isolated from snow compared with organic and inorganic substrates

Environmental Chemistry ◽

10.1071/en08055 ◽

2008 ◽

Vol 5 (6) ◽

pp. 373 ◽

Cited By ~ 37

Author(s):

Roya Mortazavi ◽

Christopher T. Hayes ◽

Parisa A. Ariya

Keyword(s):

Bacterial Species ◽

Ice Nucleation ◽

Detailed Knowledge ◽

Bacterial Populations ◽

Inorganic Aerosols ◽

Ice Nucleators ◽

Species Population ◽

Nucleation Activity ◽

Ice Nucleation Activity ◽

Snow Samples

Environmental context. Biological ice nucleators have been found to freeze water at very warm temperatures. The potential of bio-aerosols to greatly influence cloud chemistry and microphysics is becoming increasingly apparent, yet detailed knowledge of their actual role in atmospheric processes is lacking. The formation of ice in the atmosphere has significant local, regional and global influence, ranging from precipitation to cloud nucleation and thus climate. Ice nucleation tests on bacteria isolated from snow and laboratory-grown bacteria, in comparison with those of known organic and inorganic aerosols, shed light on this issue. Abstract. Ice nucleation experiments on bacteria isolated from snow as well as grown in the laboratory, in comparison with those of known organic and inorganic aerosols, examined the importance of bio-aerosols on cloud processes. Snow samples were collected from urban and suburban sites in the greater Montreal region in Canada (45°28′N, 73°45′W). Among many snow bacterial isolates, eight types of bacterial species, none belonging to known effective ice nucleators such as Pseudomonas or Erwinia genera, were identified to show an intermediate range of ice nucleation activity (–12.9 ± 1.3°C to –17.5 ± 2.8°C). Comparable results were also obtained for molten snow samples and inorganic suspensions (kaolin and montmorillonite) of buffered water solutions. The presence of organic molecules (oxalic, malonic and succinic acids) had minimal effect (<2°C) on ice nucleation. Considering experimental limitations, and drawing from observation in snow samples of a variety of bacterial populations with variable ice-nucleation ability, a shift in airborne-species population may significantly alter glaciation processes in clouds.

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Antimicrobial Activity of Non-steroidal Anti-inflammatory Drugs on Biofilm: Current Evidence and Potential for Drug Repurposing

Frontiers in Microbiology ◽

10.3389/fmicb.2021.707629 ◽

2021 ◽

Vol 12 ◽

Author(s):

Rodrigo Cuiabano Paes Leme ◽

Raquel Bandeira da Silva

Keyword(s):

Bacterial Species ◽

Drug Repurposing ◽

Current Evidence ◽

Pharmacokinetic Studies ◽

Bacterial Populations ◽

Human Pharmacokinetic ◽

Inflammatory Drugs ◽

Anti Inflammatory

It has been demonstrated that some non-steroidal anti-inflammatory drugs (NSAIDs), like acetylsalicylic acid, diclofenac, and ibuprofen, have anti-biofilm activity in concentrations found in human pharmacokinetic studies, which could fuel an interest in repurposing these well tolerated drugs as adjunctive therapies for biofilm-related infections. Here we sought to review the currently available data on the anti-biofilm activity of NSAIDs and its relevance in a clinical context. We performed a systematic literature review to identify the most commonly tested NSAIDs drugs in the last 5 years, the bacterial species that have demonstrated to be responsive to their actions, and the emergence of resistance to these molecules. We found that most studies investigating NSAIDs’ activity against biofilms were in vitro, and frequently tested non-clinical bacterial isolates, which may not adequately represent the bacterial populations that cause clinically-relevant biofilm-related infections. Furthermore, studies concerning NSAIDs and antibiotic resistance are scarce, with divergent outcomes. Although the potential to use NSAIDs to control biofilm-related infections seems to be an exciting avenue, there is a paucity of studies that tested these drugs using appropriate in vivo models of biofilm infections or in controlled human clinical trials to support their repurposing as anti-biofilm agents.

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Virulent secondary metabolites of entomopathogenic bacteria genera, Xenorhabdus and Photorhabdus, inhibit phospholipase A2 to suppress host insect immunity

10.21203/rs.3.rs-63434/v2 ◽

2020 ◽

Author(s):

Md. Mahi Imam Mollah ◽

Yonggyun Kim

Keyword(s):

Secondary Metabolites ◽

Bacterial Culture ◽

Bacterial Species ◽

Culture Broth ◽

Insect Immunity ◽

Bacterial Isolates ◽

Bacterial Pathogenicity ◽

Entomopathogenic Bacteria ◽

Organic Extracts ◽

Insecticidal Activities

Abstract Background: Xenorhabdus and Photorhabdus are entomopathogenic bacteria that cause septicemia and toxemia in insects. They produce secondary metabolites to induce host immunosuppression. Their metabolite compositions vary among bacterial species. Little is known about the relationship between metabolite compositions and the bacterial pathogenicity. The objective of this study was to compare pathogenicity and production of secondary metabolites of 14 bacterial isolates (species or strains) of Xenorhabdus and Photorhabdus. Results: All bacterial isolates exhibited insecticidal activities after hemocoelic injection to Spodoptera exigua (a lepidopteran insect) larvae, with median lethal doses ranging from 168.8 to 641.3 CFU per larva. Bacterial infection also led to immunosuppression by inhibiting eicosanoid biosynthesis. Bacterial culture broth was fractionated into four different organic extracts. All four organic extracts of each bacterial species exhibited insecticidal activities and resulted in immunosuppression. These organic extracts were subjected to GC-MS analysis which predicted 182 compounds, showing differential compositions for 14 bacteria isolates. There were positive correlations between total number of secondary metabolites produced by each bacterial culture broth and its bacterial pathogenicity based on immunosuppression and insecticidal activity. From these correlation results, 70 virulent compounds were selected from secondary metabolites of high virulent bacterial isolates by deducting those of low virulent bacterial isolates. These selected virulent compounds exhibited significant immunosuppressive activities by inhibiting eicosanoid biosynthesis. They also exhibited relatively high insecticidal activities. Conclusion: Virulence variation between Xenorhabdus and Photorhabdus is determined by their different compositions of secondary metabolites, of which PLA2 inhibitors play a crucial role.

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A Trifecta of New Insights into Ovine Footrot for Infection Drivers, Immune Response and Host Pathogen Interactions.

Infection and Immunity ◽

10.1128/iai.00270-21 ◽

2021 ◽

Author(s):

Adam M. Blanchard ◽

Ceri E. Staley ◽

Laurence Shaw ◽

Sean R Wattegedera ◽

Christina-Marie Baumbach ◽

...

Keyword(s):

Immune Response ◽

Type I Collagen ◽

Global Analysis ◽

Bacterial Species ◽

Skin Barrier ◽

Control Measures ◽

Skin Barrier Function ◽

Type I ◽

Bacterial Populations

Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industry’s biggest welfare problems. The complex aetiology of footrot makes in-situ or in-vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved, how they may interact with the host and ultimately providing a way to identify targets for future hypotheses driven investigative work. Here we present the first combined global analysis of the bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intra tissue and surface bacterial populations and the most abundant bacterial transcriptome were analysed, demonstrating footrot affected skin has a reduced diversity and increased abundances of, not only the causative bacteria Dichelobacter nodosus , but other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica . Host transcriptomics reveals a suppression of biological processes relating to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot affected interdigital skin comparted to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome which could lead to the identification of future therapeutic targets. Impact Statement Lameness in sheep is a global welfare and economic concern and footrot is the leading cause of lameness, affecting up to 70% of flocks in the U.K. Current methods for control of this disease are labour intensive and account for approximately 65% of antibiotic use in sheep farming, whilst preventative vaccines suffer from poor efficacy due to antigen competition. Our limited understanding of cofounders, such as strain variation and polymicrobial nature of infection mean new efficacious, affordable and scalable control measures are not receiving much attention. Here we examine the surface and intracellular bacterial populations and propose potential interactions with the host. Identification of these key bacterial species involved in the initiation and progression of disease and the host immune mechanisms could help form the basis of new therapies.

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Genotyping of evolving prokaryotic populations

10.7287/peerj.preprints.2449v1 ◽

2016 ◽

Author(s):

Markus Zojer ◽

Lisa N Schuster ◽

Frederik Schulz ◽

Alexander Pfundner ◽

Matthias Horn ◽

...

Keyword(s):

Experimental Evolution ◽

Bacterial Species ◽

Clinical Diagnostics ◽

Structural Variations ◽

Natural Habitats ◽

Reliable Prediction ◽

Low Frequencies ◽

Bacterial Populations ◽

Sequencing Errors ◽

Genomic Environment

Genomic heterogeneity of bacterial species is observed and studied in experimental evolution experiments, clinical diagnostics and occurs as micro-diversity of natural habitats. The challenge for genome research is to accurately capture this heterogeneity with the currently used short sequencing reads. Recent advances in NGS technologies improved the speed and coverage and thus allowed for deep sequencing of bacterial populations. This facilitates the quantitative assessment of genomic heterogeneity, including low frequent alleles or haplotypes. However, false positive variant predictions due to sequencing errors and mapping artifacts of short reads need to be prevented. We therefore created VarCap, a workflow for the reliable prediction of different types of variants even at low frequencies. In order to predict SNPs, indels and structural variations, we evaluated the sensitivity and accuracy of different software tools using synthetic read data. The results suggested that the best sensitivity could be reached by a combination of different tools. We identified possible reasons for false predictions and used this knowledge to improve the accuracy by post-filtering the predicted variants according to properties such as frequency, coverage, genomic environment/localization and co-localization with other variants. This resulted in the reliable prediction of variants above a minimum relative abundance of 2%. VarCap is designed for being routinely used within experimental evolution experiments or for clinical diagnostics. The detected variants are reported as frequencies within a vcf file and as a graphical overview of the distribution of the different variant/allele/haplotype frequencies. The source code of VarCap is available at https://github.com/ma2o/VarCap. In order to provide this workflow to a broad community, we implemeted VarCap on a Galaxy webserver (Afgan et al. 2016) , which is accessible at http://galaxy.csb.univie.ac.at.

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Heterogeneous ceramide distributions alter spatially resolved growth of Staphylococcus aureus on human stratum corneum

Journal of The Royal Society Interface ◽

10.1098/rsif.2017.0848 ◽

2018 ◽

Vol 15 (141) ◽

pp. 20170848 ◽

Cited By ~ 4

Author(s):

Joseph M. Cleary ◽

Zachary W. Lipsky ◽

Minyoung Kim ◽

Cláudia N. H. Marques ◽

Guy K. German

Keyword(s):

Staphylococcus Aureus ◽

Stratum Corneum ◽

Bacterial Growth ◽

Disease Transmission ◽

Bacterial Species ◽

Causal Link ◽

Bacterial Populations ◽

Human Stratum Corneum ◽

Spatially Resolved ◽

Skin Cleansing

Contemporary studies have revealed dramatic changes in the diversity of bacterial microbiota between healthy and diseased skin. However, the prevailing use of swabs to extract the microorganisms has meant that only population ‘snapshots’ are obtained, and all spatially resolved information of bacterial growth is lost. Here we report on the temporospatial growth of Staphylococcus aureus on the surface of the human stratum corneum (SC); the outermost layer of skin. This bacterial species dominates bacterial populations on skin with atopic dermatitis (AD). We first establish that the distribution of ceramides naturally present in the SC is heterogeneous, and correlates with the tissue's structural topography. This distribution subsequently impacts the growth of bacterial biofilms. In the SC retaining healthy ceramide concentrations, biofilms exhibit no spatial preference for growth. By contrast, a depletion of ceramides consistent with reductions known to occur with AD enables S. aureus to use the patterned network of topographical canyons as a conduit for growth. The ability of ceramides to govern bacterial growth is confirmed using a topographical skin canyon analogue coated with the ceramide subcomponent d -sphingosine. Our work appears to explain the causal link between ceramide depletion and increased S. aureus populations that is observed in AD. It may also provide insight into disease transmission as well as improving pre-operative skin cleansing techniques.

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Connecting Microbial Population Genetics with Microbial Pathogenesis Engineering Microfluidic Cell Arrays for High-throughput Interrogation of Host-Pathogen Interaction

Handbook of Research on Computational and Systems Biology ◽

10.4018/978-1-60960-491-2.ch023 ◽

2011 ◽

pp. 533-548

Author(s):

Palaniappan Sethu ◽

Kalyani Putty ◽

Yongsheng Lian ◽

Awdhesh Kalia

Keyword(s):

High Throughput ◽

Bacterial Species ◽

Human Cells ◽

Bacterial Strains ◽

Cell Array ◽

Bacterial Populations ◽

Host Pathogen Interaction ◽

Gradient Generator ◽

Molecular Events ◽

Host Pathogen

A bacterial species typically includes heterogeneous collections of genetically diverse isolates. How genetic diversity within bacterial populations influences the clinical outcome of infection remains mostly indeterminate. In part, this is due to a lack of technologies that can enable contemporaneous systems-level interrogation of host-pathogen interaction using multiple, genetically diverse bacterial strains. This chapter presents a prototype microfluidic cell array (MCA) that allows simultaneous elucidation of molecular events during infection of human cells in a semi-automated fashion. It shows that infection of human cells with up to sixteen genetically diverse bacterial isolates can be studied simultaneously. The versatility of MCAs is enhanced by incorporation of a gradient generator that allows interrogation of host-pathogen interaction under four different concentrations of any given environmental variable at the same time. Availability of high throughput MCAs should foster studies that can determine how differences in bacterial gene pools and concentration-dependent environmental variables affect the outcome of host-pathogen interaction.

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