Paleomicrobiology: Revealing Fecal Microbiomes of Ancient Indigenous Cultures

Coprolites are fossilized feces that can be used to provide information on the composition of the intestinal microbiota and, as we show, possibly on diet. We analyzed human coprolites from the Huecoid and Saladoid cultures from a settlement on Vieques Island, Puerto Rico. While more is known about the Saladoid culture, it is believed that both societies co-existed on this island approximately from 5 to 1170 AD. By extracting DNA from the coprolites, followed by metagenomic characterization, we show that both cultures can be distinguished from each other on the basis of their bacterial and fungal gut microbiomes. In addition, we show that parasite loads were heavy and also culturally distinct. Huecoid coprolites were characterized by maize and Basidiomycetes sequences, suggesting that these were important components of their diet. Saladoid coprolite samples harbored sequences associated with fish parasites, suggesting that raw fish was a substantial component of their diet. The present study shows that ancient DNA is not entirely degraded in humid, tropical environments, and that dietary and/or host genetic differences in ancient populations may be reflected in the composition of their gut microbiome. This further supports the hypothesis that the two ancient cultures studied were distinct, and that they retained distinct technological/cultural differences during an extended period of close proximity and peaceful co-existence. The two populations seemed to form the later-day Taínos, the Amerindians present at the point of Columbian contact. Importantly, our data suggest that paleomicrobiomics can be a powerful tool to assess cultural differences between ancient populations.     

Detection of Human-Derived Fecal Pollution in Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay

Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 μl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking “toolbox.”     

Microbial communities adhering to the obverse and reverse sides of an oil painting on canvas: identification and evaluation of their biodegradative potential

In this study, we investigated and compared the microbial communities adhering to the obverse and the reverse sides of an oil painting on canvas exhibiting signs of biodeterioration. Samples showing no visible damage were investigated as controls. Air samples were also analysed, in order to investigate the presence of airborne microorganisms suspended in the indoor atmosphere. The diversity of the cultivable microorganisms adhering to the surface was analysed by molecular techniques, such as RAPD analysis and gene sequencing. DGGE fingerprints derived from DNA directly extracted from canvas material in combination with clone libraries and sequencing were used to evaluate the non-cultivable fraction of the microbial communities associated with the material. By using culture-dependent methods, most of the bacterial strains were found to be common airborne, spore-forming microorganisms and belonged to the phyla Actinobacteria and Firmicutes, whereas culture-independent techniques identified sequenced clones affiliated with members of the phyla Actinobacteria and Proteobacteria. The diversity of fungi was shown to be much lower than that observed for bacteria, and only species of Penicillium spp. could be detected by cultivation techniques. The selected strategy revealed a higher microbial diversity on the obverse than on the reverse side of the painting and the near absence of actively growing microorganisms on areas showing no visible damage. Furthermore, enzymatic activity tests revealed that the most widespread activities involved in biodeterioration were esterase and esterase lipase among the isolated bacterial strains, and esterase and N-acetyl-β-glucosaminidase among fungi strains.     

Functional Screening of a Metagenomic Library Reveals Operons Responsible for Enhanced Intestinal Colonization by Gut Commensal Microbes

Evidence suggests that gut microbes colonize the mammalian intestine through propagation as an adhesive microbial community. A bacterial artificial chromosome (BAC) library of murine bowel microbiota DNA in the surrogate host Escherichia coli DH10B was screened for enhanced adherence capability. Two out of 5,472 DH10B clones, 10G6 and 25G1, exhibited enhanced capabilities to adhere to inanimate surfaces in functional screens. DNA segments inserted into the 10G6 and 25G1 clones were 52 and 41 kb and included 47 and 41 protein-coding open reading frames (ORFs), respectively. DNA sequence alignments, tetranucleotide frequency, and codon usage analysis strongly suggest that these two DNA fragments are derived from species belonging to the genus Bacteroides. Consistent with this finding, a large portion of the predicted gene products were highly homologous to those of Bacteroides spp. Transposon mutagenesis and subsequent experiments that involved heterologous expression identified two operons associated with enhanced adherence. E. coli strains transformed with the 10a or 25b operon adhered to the surface of intestinal epithelium and colonized the mouse intestine more vigorously than did the control strain. This study has revealed the genetic determinants of unknown commensals (probably resembling Bacteroides species) that enhance the ability of the bacteria to colonize the murine bowel.     

Chlorophenol Production by Anaerobic Microorganisms: Transformation of a Biogenic Chlorinated Hydroquinone Metabolite

Chlorinated hydroquinones of biological origin are fully dechlorinated to 1,4-dihydroquinone by anaerobic bacteria such as Desulfitobacterium spp. (C. E. Milliken, G. P. Meier, J. E. M. Watts, K. R. Sowers, and H. D. May, Appl. Environ. Microbiol. 70:385-392, 2004). In the present study, mixed microbial communities from Baltimore Harbor sediment and a pure culture of Desulfitobacterium sp. strain PCE1 were discovered to demethylate, reductively dehydroxylate, and dechlorinate chlorinated hydroquinones into chlorophenols. Mixed microbial cultures from a freshwater source and several other desulfitobacteria in pure culture did not perform these reactions. Desulfitobacterium sp. strain PCE1 degraded 2,3,5,6-tetrachloro-4-methoxyphenol, a metabolite of basidiomycete fungi, to 2,3,5,6-tetrachlorophenol and 2,3,5-trichlorophenol, recalcitrant compounds that are primarily synthesized anthropogenically.     

Insights about echinostomiasis by paleomolecular diagnosis

Echinostomiasis is a zoonosis caused by intestinal trematodes and transmitted by the ingestion of mollusks, crustaceans, fish, amphibians, and reptiles, either raw or poorly cooked. Today human infection is endemic in Southeast Asia and the Far East, but has been reported more recently in other regions of the world. Interestingly eggs identified as Echinostoma sp. were found in coprolites from a mummified body human in Brazil, dated 560 ± 40 BP (before present). However, the specific diagnosis based on morphology of the eggs has not been resolved at the species level. As a follow-up to the previous finding, the current study now aims to standardize the methodology for molecular diagnosis and apply it to the coprolite, using current Echinostoma paraensei-positive feces as the reference, and also the same fecal material dried in a stove as an experimental coprolite model. Isolated eggs of E. paraensei and adult worm were included to verify the sensibility and as positive control, respectively. An adult worm of E. luisreyi was used for comparison. PCR using primers in-house for ITS1 region (126 bp) and cox1 (123 bp) of Echinostoma spp. and subsequent nucleotide sequencing were performed. This is the first molecular paleoparasitological diagnosis for echinostomiasis. The methodology was able to amplify specific DNA fragments for the genus Echinostoma sp. in all samples: adult worm, feces, and a single egg of the parasite, in both the experimental coprolite and archaeological sample. Additionally we observed that ancient DNA can also be retrieved without rehydrating the material. The nucleotide sequences from E. paraensei and E. luisreyi are very similar in the fragment analyzed that difficult the differentiation these species, but DNA sequence analysis recovered in the parasite found in the mummy showed more similarity with the species E. paraensei.     

Molecular Analysis of Shower Curtain Biofilm Microbes

Households provide environments that encourage the formation of microbial communities, often as biofilms. Such biofilms constitute potential reservoirs for pathogens, particularly for immune-compromised individuals. One household environment that potentially accumulates microbial biofilms is that provided by vinyl shower curtains. Over time, vinyl shower curtains accumulate films, commonly referred to as “soap scum,” which microscopy reveals are constituted of lush microbial biofilms. To determine the kinds of microbes that constitute shower curtain biofilms and thereby to identify potential opportunistic pathogens, we conducted an analysis of rRNA genes obtained by PCR from four vinyl shower curtains from different households. Each of the shower curtain communities was highly complex. No sequence was identical to one in the databases, and no identical sequences were encountered in the different communities. However, the sequences generally represented similar phylogenetic kinds of organisms. Particularly abundant sequences represented members of the α-group of proteobacteria, mainly Sphingomonas spp. and Methylobacterium spp. Both of these genera are known to include opportunistic pathogens, and several of the sequences obtained from the environmental DNA samples were closely related to known pathogens. Such organisms have also been linked to biofilm formation associated with water reservoirs and conduits. In addition, the study detected many other kinds of organisms at lower abundances. These results show that shower curtains are a potential source of opportunistic pathogens associated with biofilms. Frequent cleaning or disposal of shower curtains is indicated, particularly in households with immune-compromised individuals.     

Detection of Genetic Markers of Fecal Indicator Bacteria in Lake Michigan and Determination of Their Relationship to Escherichia coli Densities Using Standard Microbiological Methods

Lake Michigan surface waters impacted by fecal pollution were assessed to determine the occurrence of genetic markers for Bacteroides and Escherichia coli. Initial experiments with sewage treatment plant influent demonstrated that total Bacteroides spp. could be detected by PCR in a 25- to 125-fold-higher dilution series than E. coli and human-specific Bacteroides spp., which were both found in similar dilution ranges. The limit of detection for the human-specific genetic marker ranged from 0.2 CFU/100 ml to 82 CFU/100 ml culturable E. coli for four wastewater treatment plants in urban and rural areas. The spatial and temporal distributions of these markers were assessed following major rain events that introduced urban storm water, agricultural runoff, and sewage overflows into Lake Michigan. Bacteroides spp. were detected in all of these samples by PCR, including those with <1 CFU/100 ml E. coli. Human-specific Bacteroides spp. were detected as far as 2 km into Lake Michigan during sewage overflow events, with variable detection 1 to 9 days postoverflow, whereas the cow-specific Bacteroides spp. were detected in only highly contaminated samples near the river outflow. Lake Michigan beaches were also assessed throughout the summer season for the same markers. Bacteroides spp. were detected in all beach samples, including 28 of the 74 samples that did not exceed 235 CFU/100 ml of E. coli. Human-specific Bacteroides spp. were detected at three of the seven beaches; one of the sites demonstrating positive results was sampled during a reported sewage overflow, but E. coli levels were below 235 CFU/100 ml. This study demonstrates the usefulness of non-culture-based microbial-source tracking approaches and the prevalence of these genetic markers in the Great Lakes, including freshwater coastal beaches.     

Specific Microbial Communities Associate with the Rhizosphere of Welwitschia mirabilis,a Living Fossil

Welwitschia mirabilis is an ancient and rare plant distributed along the western coast of Namibia and Angola. Several aspects of Welwitschia biology and ecology have been investigated, but very little is known about the microbial communities associated with this plant. This study reports on the bacterial and fungal communities inhabiting the rhizosphere of W. mirabilis and the surrounding bulk soil. Rhizosphere communities were dominated by sequences of Alphaproteobacteria and Euromycetes, while Actinobacteria, Alphaproteobacteria, and fungi of the class Dothideomycetes jointly dominated bulk soil communities. Although microbial communities within the rhizosphere and soil samples were highly variable, very few “species” (OTUs defined at a 97% identity cut-off) were shared between these two environments. There was a small ‘core’ rhizosphere bacterial community (formed by Nitratireductor, Steroidobacter, Pseudonocardia and three Phylobacteriaceae) that together with Rhizophagus, an arbuscular mycorrhizal fungus, and other putative plant growth-promoting microbes may interact synergistically to promote Welwitschia growth.     

Dynamics of Bacterial and Fungal Communities on Decaying Salt Marsh Grass

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated microbial community. Here we describe the bacterial and fungal assemblages associated with two decomposition stages of Spartina alterniflora detritus in a productive southeastern U.S. salt marsh. 16S rRNA genes and 18S-to-28S internal transcribed spacer (ITS) regions were used to target the bacterial and ascomycete fungal communities, respectively, based on DNA sequence analysis of isolates and environmental clones and by using community fingerprinting based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Seven major bacterial taxa (six affiliated with the α-Proteobacteria and one with the Cytophagales) and four major fungal taxa were identified over five sample dates spanning 13 months. Fungal terminal restriction fragments (T-RFs) were informative at the species level; however, bacterial T-RFs frequently comprised a number of related genera. Amplicon abundances indicated that the salt marsh saprophyte communities have little-to-moderate variability spatially or with decomposition stage, but considerable variability temporally. However, the temporal variability could not be readily explained by either successional shifts or simple relationships with environmental factors. Significant correlations in abundance (both positive and negative) were found among dominant fungal and bacterial taxa that possibly indicate ecological interactions between decomposer organisms. Most associations involved one of four microbial taxa: two groups of bacteria affiliated with the α-Proteobacteria and two ascomycete fungi (Phaeosphaeria spartinicola and environmental isolate “4clt”).     

Paleocene Thalassinidea colonization in deep-sea environment and the coprolite Palaxius osaensis n. ichnosp. in Southern Costa Rica

Palaxius osaensis n. ichnosp., a new ichnospecies of crustacean coprolite is described. The coprolite is preserved in a 200-m thick Paleocene sequence in Southern Costa Rica that is largely dominated by pillow basalts. The studied sample is part of a seamount formed in the Pacific Ocean that was accreted to the Central American isthmus during the Eocene. The absence of lava vesicles, shallow-water deposits, and detrital sediments in the section suggest that the coprolites were deposited in a deep environment during the first stage of the development of the seamount. This represents one of the deepest occurrences of Thalassinidea coprolites reported in the literature and indicates that the producers of the coprolites, presumably some shrimps, developed the aptitude to colonize abyssal environments at least since Early Tertiary. The crustacean coprolites were encountered at a site which apparently lacked a food supply, although hydrothermal processes are believed to have provided the opportunity for a chemotrophic community to develop on the deepest part of the seamount. P. osaensis n. ichnosp. is also found in Colombia in late Cretaceous shallow-water sediments that notably contain Palaxius caucaensis coprolites (Micropaleontology 41 (1995) 85-88). Occurrences of P. osaensis n. ichnosp. deposited at both shallow and deep levels may possibly be related to an aptitude of some thalassinid organisms to have developed in various biotopes during the late Cretaceous-Paleocene.     

Possible Origins of CTnBST, a Conjugative Transposon Found Recently in a Human Colonic Bacteroides Strain

A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species.     

Comparison of Acid Mine Drainage Microbial Communities in Physically and Geochemically Distinct Ecosystems

This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of ~0.5 to ~1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. “Ferroplasma” spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength. Leptospirillum spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured Leptospirillum sp. dominated. Sulfobacillus spp. were detected as a prominent inhabitant in warmer (~43°C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.     

Identification of unique microbiomes associated with harmful algal blooms caused by Alexandrium fundyense and Dinophysis acuminata

Biotic interactions dominate plankton communities, yet the microbial consortia associated with harmful algal blooms (HABs) have not been well-described. Here, high-throughput amplicon sequencing of ribosomal genes was used to quantify the dynamics of bacterial (16S) and phytoplankton assemblages (18S) associated with blooms and cultures of two harmful algae, Alexandrium fundyense and Dinophysis acuminata. Experiments were performed to assess changes in natural bacterial and phytoplankton communities in response to the filtrate from cultures of these two harmful algae. Analysis of prokaryotic sequences from ecosystems, experiments, and cultures revealed statistically unique bacterial associations with each HAB. The dinoflagellate, Alexandrium, was strongly associated with multiple genera of Flavobacteria including Owenweeksia spp., Maribacter spp., and individuals within the NS5 marine group. While Flavobacteria also dominated Dinophysis-associated communities, the relative abundance of Alteromonadales bacteria strongly co-varied with Dinophysis abundances during blooms and Ulvibacter spp. (Flavobacteriales) and Arenicella spp. (Gammaproteobacteria) were associated with cells in culture. Eukaryotic sequencing facilitated the discovery of the endosymbiotic, parasitic dinoflagellate, Amoebophrya spp., that had not been regionally described but represented up to 17% of sequences during Alexandrium blooms. The presence of Alexandrium in field samples and in experiments significantly altered the relative abundances of bacterial and phytoplankton by both suppressing and promoting different taxa, while this effect was weaker in Dinophysis. Experiments specifically revealed a negative feedback loop during blooms whereby Alexandrium filtrate promoted the abundance of the parasite, Amoebophrya spp. Collectively, this study demonstrates that HABs formed by Alexandrium and Dinophysis harbor unique prokaryotic and eukaryotic microbiomes that are likely to, in turn, influence the dynamics of these HABs.     

Microbiota and food residues including possible evidence of pre‐mammalian hair in Upper Permian coprolites from Russia

Coprolites (fossil faeces) provide direct evidence on the diet of its producer and unique insights on ancient food webs and ecosystems. We describe the contents of seven coprolites, collected from the Late Permian Vyazniki site of the European part of Russia. Two coprolite morphotypes (A, B) contain remains of putative bacteria, cyanobacteria, fungi, protists, invertebrate eggs, arthropod elements, undigested bone and tooth fragments, fish scales and elongated hair‐like structures with hollow interiors. Content, size and shape of the coprolites together with the associated body fossil record suggest that the most probable scat‐producers were carnivorous tetrapods; the bone‐rich morphotype A reveals short food retention time and a fast metabolism and is therefore assigned to therapsid carnivores whereas morphotype B with rarer and degraded bones are assigned to archosauromorphs or other non‐therapsid carnivores. The general coprolite matrix contains abundant micron‐sized spheres and thin‐walled vesicles which are interpreted as oxide and phosphatic pseudomorphs after microbial cells. From analyses of the undigested bones, we infer that they represent remains of actinopterygian fish, a therapsid and unrecognizable parts of amphibians and/or reptiles. Additionally, hair‐like structures found in one coprolite specimen occur as diagenetically altered (oxide‐replaced) structures and moulds (or partly as pseudomorphs) in a microcrystalline carbonate‐fluoride‐bearing calcium phosphate. This suggests that the latest Permian therapsids probably were equipped with hair‐like integument or hairsuit. If true, this is by far the oldest evidence of this mammalian character in the stem group of mammals.     

Mucosa-Associated Bacterial Diversity in Necrotizing Enterocolitis

Background

Previous studies of infant fecal samples have failed to clarify the role of gut bacteria in the pathogenesis of NEC. We sought to characterize bacterial communities within intestinal tissue resected from infants with and without NEC.

Methods

26 intestinal samples were resected from 19 infants, including 16 NEC samples and 10 non-NEC samples. Bacterial 16S rRNA gene sequences were amplified and sequenced. Analysis allowed for taxonomic identification, and quantitative PCR was used to quantify the bacterial load within samples.

Results

NEC samples generally contained an increased total burden of bacteria. NEC and non-NEC sample sets were both marked by high inter-individual variability and an abundance of opportunistic pathogens. There was no statistically significant distinction between the composition of NEC and non-NEC microbial communities. K-means clustering enabled us to identify several stable clusters, including clusters of NEC and midgut volvulus samples enriched with Clostridium and Bacteroides. Another cluster containing both NEC and non-NEC samples was marked by an abundance of Enterobacteriaceae and decreased diversity among NEC samples.

Conclusions

The results indicate that NEC is a disease without a uniform pattern of microbial colonization, but that NEC is associated with an abundance of strict anaerobes and a decrease in community diversity.     

Microbial Biofilm Formation and Contamination of Dental-Unit Water Systems in General Dental Practice

Dental-unit water systems (DUWS) harbor bacterial biofilms, which may serve as a haven for pathogens. The aim of this study was to investigate the microbial load of water from DUWS in general dental practices and the biofouling of DUWS tubing. Water and tube samples were taken from 55 dental surgeries in southwestern England. Contamination was determined by viable counts on environmentally selective, clinically selective, and pathogen-selective media, and biofouling was determined by using microscopic and image analysis techniques. Microbial loading ranged from 500 to 10⁵ CFU · ml⁻¹; in 95% of DUWS water samples, it exceeded European Union drinking water guidelines and in 83% it exceeded American Dental Association DUWS standards. Among visible bacteria, 68% were viable by BacLight staining, but only 5% of this “viable by BacLight” fraction produced colonies on agar plates. Legionella pneumophila, Mycobacterium spp., Candida spp., and Pseudomonas spp. were detected in one, five, two, and nine different surgeries, respectively. Presumptive oral streptococci and Fusobacterium spp. were detected in four and one surgeries, respectively, suggesting back siphonage and failure of antiretraction devices. Hepatitis B virus was never detected. Decontamination strategies (5 of 55 surgeries) significantly reduced biofilm coverage but significantly increased microbial numbers in the water phase (in both cases, P < 0.05). Microbial loads were not significantly different in DUWS fed with soft, hard, deionized, or distilled water or in different DUWS (main, tank, or bottle fed). Microbiologically, no DUWS can be considered “cleaner” than others. DUWS deliver water to patients with microbial levels exceeding those considered safe for drinking water.     

Gut Microbiome of an 11th Century A.D. Pre-Columbian Andean Mummy

The process of natural mummification is a rare and unique process from which little is known about the resulting microbial community structure. In the present study, we characterized the microbiome of paleofeces, and ascending, transverse and descending colon of an 11^th century A.D. pre-Columbian Andean mummy by 16S rRNA gene high-throughput sequencing and metagenomics. Firmicutes were the most abundant bacterial group, with Clostridium spp. comprising up to 96.2% of the mummified gut, while Turicibacter spp. represented 89.2% of the bacteria identified in the paleofeces. Microbiome profile of the paleofeces was unique when compared to previously characterized coprolites that did not undergo natural mummification. We identified DNA sequences homologous to Clostridium botulinum, Trypanosoma cruzi and human papillomaviruses (HPVs). Unexpectedly, putative antibiotic-resistance genes including beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters, were also identified. The presence of putative antibiotic-resistance genes suggests that resistance may not necessarily be associated with a selective pressure of antibiotics or contact with European cultures. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses.     

Detritus-Dependent Development of the Microbial Community in an Experimental System: Qualitative Analysis by Denaturing Gradient Gel Electrophoresis

Correlations between the biomass of phytoplankton and the biomass of bacteria and between the biomass of bacteria and the biomass of protozoans suggest that there is coupling between these compartments of the “microbial loop.” To investigate this coupling on the species level, bacteria and protozoans from untreated lake water inocula were allowed to grow on detritus of the green alga Ankistrodesmus falcatus or the cyanobacterium Oscillatoria limnetica in continuous-flow systems for 1 month. Denaturing gradient gel electrophoresis (DGGE) of the 16S and 18S rRNA genes was used to monitor the development of the bacterial community structure and the eukaryotic community structure, respectively. Nonmetric multidimensional scaling of the DGGE profiles revealed the changes in the microbial community structure. This analysis showed that significantly different bacterial communities developed on the green algal detritus and on the cyanobacterial detritus. Although similar results were obtained for the eukaryotic communities, the differences were not significant. Hence, our findings indicate that the origin of detritus can affect the structure of at least the bacterial community. A phylogenetic analysis of 20 18S ribosomal DNA clones that were isolated from the continuous cultures revealed that many sequences were related to the sequences of bacterivorous protozoans (members of the Ciliophora, Rhizopoda, Amoeba, and Kinetoplastida). One clone grouped in a recently established clade whose previously described members are all parasites. The affiliations of about 20% of the clones could not be determined.     

Microbial Communities Associated with the Larval Gut and Eggs of the Western Corn Rootworm

Background

The western corn rootworm (WCR) is one of the economically most important pests of maize. A better understanding of microbial communities associated with guts and eggs of the WCR is required in order to develop new pest control strategies, and to assess the potential role of the WCR in the dissemination of microorganisms, e.g., mycotoxin-producing fungi.

Methodology/Principal Findings

Total community (TC) DNA was extracted from maize rhizosphere, WCR eggs, and guts of larvae feeding on maize roots grown in three different soil types. Denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene and ITS fragments, PCR-amplified from TC DNA, were used to investigate the fungal and bacterial communities, respectively. Microorganisms in the WCR gut were not influenced by the soil type. Dominant fungal populations in the gut were affiliated to Fusarium spp., while Wolbachia was the most abundant bacterial genus. Identical ribosomal sequences from gut and egg samples confirmed a transovarial transmission of Wolbachia sp. Betaproteobacterial DGGE indicated a stable association of Herbaspirillum sp. with the WCR gut. Dominant egg-associated microorganisms were the bacterium Wolbachia sp. and the fungus Mortierella gamsii.

Conclusion/Significance

The soil type-independent composition of the microbial communities in the WCR gut and the dominance of only a few microbial populations suggested either a highly selective environment in the gut lumen or a high abundance of intracellular microorganisms in the gut epithelium. The dominance of Fusarium species in the guts indicated WCR larvae as vectors of mycotoxin-producing fungi. The stable association of Herbaspirillum sp. with WCR gut systems and the absence of corresponding sequences in WCR eggs suggested that this bacterium was postnatally acquired from the environment. The present study provided new insights into the microbial communities associated with larval guts and eggs of the WCR. However, their biological role remains to be explored.