Natural Bacterial Populations Research Articles

Biofilms: How Structure Emerges from Conflict

Natural bacterial populations often live on surfaces in complex communities called biofilms. These stress-resistant groups are often thought to result from cooperative interactions between disparate species, but recent experiments argue that biofilms are primarily a protective response to competition.

Bacteriophages limit the existence conditions for conjugative plasmids.

ABSTRACTBacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome.

Alarmingly High Segregation Frequencies of Quinolone Resistance Alleles within Human and Animal Microbiomes Are Not Explained by Direct Clinical Antibiotic Exposure.

Antibiotic resistance poses a major threat to human health. It is therefore important to characterize the frequency of resistance within natural bacterial environments. Many studies have focused on characterizing the frequencies with which horizontally acquired resistance genes segregate within natural bacterial populations. Yet, very little is currently understood regarding the frequency of segregation of resistance alleles occurring within the housekeeping targets of antibiotics. We surveyed a large number of metagenomic datasets extracted from a large variety of host-associated and non host-associated environments for such alleles conferring resistance to three groups of broad spectrum antibiotics: streptomycin, rifamycins, and quinolones. We find notable segregation frequencies of resistance alleles occurring within the target genes of each of the three antibiotics, with quinolone resistance alleles being the most frequent and rifamycin resistance alleles being the least frequent. Resistance allele frequencies varied greatly between different phyla and as a function of environment. The frequency of quinolone resistance alleles was especially high within host-associated environments, where it averaged an alarming ∼40%. Within host-associated environments, resistance to quinolones was most often conferred by a specific resistance allele. High frequencies of quinolone resistance alleles were also found within hosts that were not directly treated with antibiotics. Therefore, the high segregation frequency of quinolone resistance alleles occurring within the housekeeping targets of antibiotics in host-associated environments does not seem to be the sole result of clinical antibiotic usage.

Epidemic Clones, Oceanic Gene Pools, and Eco-LD in the Free Living Marine Pathogen Vibrio parahaemolyticus.

We investigated global patterns of variation in 157 whole-genome sequences of Vibrio parahaemolyticus, a free-living and seafood associated marine bacterium. Pandemic clones, responsible for recent outbreaks of gastroenteritis in humans, have spread globally. However, there are oceanic gene pools, one located in the oceans surrounding Asia and another in the Mexican Gulf. Frequent recombination means that most isolates have acquired the genetic profile of their current location. We investigated the genetic structure in the Asian gene pool by calculating the effective population size in two different ways. Under standard neutral models, the two estimates should give similar answers but we found a 27-fold difference. We propose that this discrepancy is caused by the subdivision of the species into a hundred or more ecotypes which are maintained stably in the population. To investigate the genetic factors involved, we used 51 unrelated isolates to conduct a genome-wide scan for epistatically interacting loci. We found a single example of strong epistasis between distant genome regions. A majority of strains had a type VI secretion system associated with bacterial killing. The remaining strains had genes associated with biofilm formation and regulated by cyclic dimeric GMP signaling. All strains had one or other of the two systems and none of isolate had complete complements of both systems, although several strains had remnants. Further "top down" analysis of patterns of linkage disequilibrium within frequently recombining species will allow a detailed understanding of how selection acts to structure the pattern of variation within natural bacterial populations.

Distinctive proteolytic activity of cell envelope proteinase of Lactobacillus helveticus isolated from airag, a traditional Mongolian fermented mare's milk

Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35°C for all the Mongolian strains, which is lower than those for the reference strains (JCM 1554 and JCM 1120T) isolated from European cheeses. All of the strains had a prtH1-like gene encoding a variant type of cell envelope proteinase (CEP). The CEP amino acid sequence in Snow Brand Typeculture (SBT) 11087 isolated from airag shared 71% identity with PrtH of L. helveticus CNRZ32 (AAD50643.1) but 98% identity with PrtH of Lactobacillus kefiranofaciens ZW3 (AEG40278.1) isolated from a traditional fermented milk in Tibet. The proteolytic activities of the CEP from SBT11087 on artificial substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and pure casein were measured using an intact-cell degradation assay. The activity of the CEP from SBT11087 was observed to be weak and exhibited a lower optimal temperature (40°C) than those from the reference strains (45–50°C). The specificity of the SBT11087 CEP for αS1-casein was typical of the CEPs previously reported in L. helveticus, as determined through the degradation profiles obtained through gel electrophoresis and mass spectrometry analyses. In contrast, the degradation profile of β-casein revealed that the CEP of SBT11087 primarily hydrolyzes its C-terminal domain and hydrolyzed nine of the 16 cleavage sites shared among the CEPs of other L. helveticus strains. Thus, the CEP of SBT11087 is distinct from those from previously reported L. helveticus strains in terms of its optimal temperature and its degradation of β-casein. Therefore, the Mongolian L. helveticus strains differ from other strains of the species in different collections and are specifically suited for the natural lactic acid bacterial population in airag.

Agrochemicals indirectly increase survival of E. coli O157:H7 and indicator bacteria by reducing ecosystem services.

Storm water and agricultural runoff frequently contain agrochemicals, fecal indicator bacteria (FIB), and zoonotic pathogens. Entry of such contaminants into aquatic ecosystems may affect ecology and human health. This study tested the hypothesis that the herbicide atrazine and the fungicide chlorothalonil indirectly affect the survival of FIB (Escherichia coli and Enterococcus faecalis) and a pathogen (E. coli O157:H7) by altering densities of protozoan predators or by altering competition from autochthonous bacteria. Streptomycin-resistant E. coli, En. faecalis, and E. coli O157:H7 were added to microcosms composed of Florida river water containing natural protozoan and bacterial populations. FIB, pathogen, and protozoan densities were monitored over six days. Known metabolic inhibitors, cycloheximide and streptomycin, were used to inhibit autochthonous protozoa or bacteria, respectively. The inhibitors made it possible to isolate the effects of predation or competition on survival of allochthonous bacteria, and each treatment increased the survival of FIB and pathogens. Chlorothalonil's effect was similar to that of cycloheximide, significantly reducing protozoan densities and elevating densities of FIB and pathogens relative to the control. Atrazine treatment did not affect protozoan densities, but, through an effect on competition, resulted in significantly greater densities of En. faecalis and E. coli O157:H7. Hence, by reducing predaceous protozoa and bacterial competitors that facilitate purifying water bodies of FIBs and human pathogens, chlorothalonil and atrazine indirectly diminished an ecosystem service of fresh water.

Nitrification of anaerobic digester effluent for nitrogen management at swine farms

Anaerobic digester effluent collected from a swine farm was nitrified in lab-scale bioreactors mimicking an innovative manure management system to understand factors relevant to a successful start-up. The effects of digester effluent strength, aeration time, nitrifying sludge inoculation, and initial pH control on the startup of the nitrification system were investigated. The results showed that initial NH4+-N concentrations higher than 800mgL−1 could severely inhibit the onset of nitrification if relying on native bacterial populations. When aeration time was increased from 4 to 12hd−1, ammonium oxidation occurred earlier, but nitrite oxidation was delayed. However, the delay was not observed when bioaugmentation with nitrifying sludge was conducted. Initial addition of acid for pH control was unsuccessful as initial depletion of alkalinity eventually resulted in self-inhibition of nitrification cause by decreasing pH over time. Overall, these results provide guidance on how to effectively start large-scale innovative animal waste treatment systems.

The ecological roles of Bacillus thuringiensis within phyllosphere environments

Bacillus thuringiensis (Bt) is one of the most used bio-control agents to control plant insects, but little is known about its effect on the microbial population and communities on plant leaves. With the culture dependent method, it has been observed that the dynamics of Bt within the phyllosphere varied dependent on both the doses of Bt sprayed on the leaves and the plant species, however, Bt’s population size kept stable at about 1000cfug−1 after 15d since inoculation. By comparing the bacterial abundances and community structures within the phyllosphere of three plant species, we confirmed that Bt at the doses of 1.5×107 and 1.5×109 cfumL−1 respectively did not significantly influence the natural bacterial population size on the leaf surfaces based on culture dependent assay. However, based on culture independent denaturing gradient gel electrophoresis (DGGE), Shannon–Wiener index (H′) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analysis, Bt has a significant influence on the bacterial communities within the phyllosphere of amaranth and cotton, but not rice. These results indicate that Bt exhibits different behaviors and ecological roles on the microbial diversity within the phyllosphere, and its environmental safety has to be concerned and evaluated in the future.

Glyphosate persistence in seawater

Glyphosate is one of the most widely applied herbicides globally but its persistence in seawater has not been reported. Here we quantify the biodegradation of glyphosate using standard “simulation” flask tests with native bacterial populations and coastal seawater from the Great Barrier Reef. The half-life for glyphosate at 25°C in low-light was 47days, extending to 267days in the dark at 25°C and 315days in the dark at 31°C, which is the longest persistence reported for this herbicide. AMPA, the microbial transformation product of glyphosate, was detected under all conditions, confirming that degradation was mediated by the native microbial community. This study demonstrates glyphosate is moderately persistent in the marine water under low light conditions and is highly persistent in the dark. Little degradation would be expected during flood plumes in the tropics, which could potentially deliver dissolved and sediment-bound glyphosate far from shore.

Heavy oil exposure increases viral production in natural marine bacterial populations

This study examined whether heavy oil (HO) increases viral production and how that change may affect the marine bacterial community. The addition of a relatively low concentration (10 μg/mL) of HO to seawater resulted in the highest degree of viral lysis. Although the composition of the bacterial community did not change upon the viral lysis in terms of the taxa present, the relative abundance of the γ-Proteobacteria family Alteromonadaceae decreased (by 10 %) after the HO exposure, implying that the selective lysis by viruses and induced prophages may be responsible for changes in the composition of the bacterial community.

English

  Natural products have been an important historical source of therapeutic agents. Microorganisms are major source of bioactive natural products, and several microbial products including antibiotics, anti-inflammatory, anti-tumour, immunosuppressants and others are currently used as therapeutic agents for human and domestic animals. Most of these products were obtained from cultured environmental microorganisms. However, it is widely accepted that a very large majority of the microorganisms present in natural environments are not readily cultured under laboratory conditions, and therefore are not accessible for drug discovery. Metagenomics is a recent culture-independent approach that has been developed to access the collective genomes of natural bacterial populations. It enables discovery of the diverse biosynthetic pathways encoded by diverse microbial assemblages that are known to be present in the environment but not-yet cultured. Recently, several new bioactive molecules and proteins have been discovered using a metagenomic approach. This review highlights the recent methodologies, limitations, and applications of metagenomics for the discovery of new drugs. Moreover, it shows how a multidisciplinary approach combining metagenomics with other technologies can expedite and revolutionize drug discovery from diverse environmental microorganisms.   Key words: Microbial diversity, metagenomics, natural products, drug discovery, microbial ecology

Bacterial response to dynamic metal concentrations in the surface sediments of a solar saltern (Goa, India)

The Ribandar solar saltern, situated adjacent to the Mandovi estuary is influenced by the barge transport of ferromanganese ore to the Mormugao harbour (Arabian Sea). The current study focuses on the distribution of metals and related heterotrophic bacterial populations in the surface sediments (0-10 cm) of the Ribandar salterns (Goa, India) during the salt-making (January to May) and non salt-making seasons (August and November). The concentrations of heavy metals in the sediments ranged from 17.2 ± 2.8 to 26.3 ± 6.7 % Fe; 0.6 ± 0.2 to 0.9 ± 0.2 % Mn; 27.6 ± 7.3 to 51 ± 8.3 ppm Ni; 28.4 ± 8.9 to 35.2 ± 10.6 ppm Co; 44 ± 21.6 to 62.8 ± 23.6 ppm Zn; 0.1 ± 0.01 ppm Cd and 1.7 ± 0.1 to 2.6 ± 0.7 ppm Pb and were much higher than those reported at the same site in a previous study by Kerkar (2004). Hg concentrations were below detection limits. In general, computation of "geoaccumulation index" revealed the sediments as 'uncontaminated to moderately contaminated' with Fe, Mn, Ni, Co, Pb and Zn during the salt-making season. The abundance of metal-tolerant bacteria was comparatively restricted to the salt-making season and was higher than the non salt-making season. Fe-, Mn-, Ni-, Co- and Pb- (200 ppm) tolerant bacteria were retrieved and restricted to the surface sediments (0-5 cm), Cd and Fe being the two most regulatory elements governing bacterial populations in the non salt-making season. However, during the salt-making season, the concentration of Zn was found to be pivotal in regulating the counts of Fe-, Mn- and Ni-tolerant bacteria. In general, the strength of correlation of metals and microbes was higher in the non salt-making season as compared to the salt-making season. This would probably indicate metal-induced limitations in microbial populations in the non salt-making season and the absence of this effect during the salt-making season. In this study, we test the hypothesis that solar salterns behave as ecological sinks with a potential to transform native bacterial populations to metal-resistant strains, in relation to the dynamic changes in the surrounding metal concentrations.

The Dynamic Cultivation System: A new method for the detection of temporal shifts in microbial community structure in clay

A major problem in the analysis of natural bacterial populations is that probably less than 1% of species were so far cultured and described. In order to overcome this disadvantage, culture independent DNA based methods, such as comparison of PCR-amplified 16S rRNA genes, are used for the description of the bacterial diversity and the identification of various species. Unfortunately, each molecule of DNA, including those of dead organisms and allochthonous genomes, can be detected by PCR-based molecular fingerprint techniques within a sample besides the DNA of native organisms. Hence, an adequate image of the actually active population composition cannot be projected with these methods. An additional challenge is the extraction of DNA from charged particles and the presence of inhibitors for downstream molecular analyses. This study reports the results of a new cultivation approach that allows to overcome these problems and to obtain almost total environmental DNA of microorganisms for subsequent genetic DNA fingerprint techniques.This new approach, the Dynamic Cultivation System (DCS) is characterized by special nutrient availability to simulate the bacterial natural habitat and the cell–cell interaction for various processes, such as collective degradation of compounds.The DCS was applied in combination with the genetic fingerprint technique Denaturing Gradient Gel Electrophoresis (DGGE) to study changes in the population composition during an alteration process of clay. The results indicate a changing bacterial diversity over time which seems to correlate to the changing pH in the clay samples in situ. This effect was most evident after cultivation on the DCS.

Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites

A single gram of soil is predicted to contain thousands of unique bacterial species. The majority of these species remain recalcitrant to standard culture methods, prohibiting their use as sources of unique bioactive small molecules. The cloning and analysis of DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means of exploring the biosynthetic capacity of natural bacterial populations. Environmental DNA libraries contain large reservoirs of bacterial genetic diversity from which new secondary metabolite gene clusters can be systematically recovered and studied. The identification and heterologous expression of type II polyketide synthase-containing eDNA clones is reported here. Functional analysis of three soil DNA-derived polyketide synthase systems in Streptomyces albus revealed diverse metabolites belonging to well-known, rare, and previously uncharacterized structural families. The first of these systems is predicted to encode the production of the known antibiotic landomycin E. The second was found to encode the production of a metabolite with a previously uncharacterized pentacyclic ring system. The third was found to encode the production of unique KB-3346-5 derivatives, which show activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These results, together with those of other small-molecule-directed metagenomic studies, suggest that culture-independent approaches are capable of accessing biosynthetic diversity that has not yet been extensively explored using culture-based methods. The large-scale functional screening of eDNA clones should be a productive strategy for generating structurally previously uncharacterized chemical entities for use in future drug development efforts.

The Binding of Triclosan to SmeT, the Repressor of the Multidrug Efflux Pump SmeDEF, Induces Antibiotic Resistance in Stenotrophomonas maltophilia

The wide utilization of biocides poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that biocides can select, at least in laboratory experiments, antibiotic resistant bacteria. This situation has raised concerns, not just on scientists and clinicians, but also on regulatory agencies, which are demanding studies on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases and on human health. In the present article, we explored the possibility that the widely used biocide triclosan might induce antibiotic resistance using as a model the opportunistic pathogen Stenotrophomonas maltophilia. Biochemical, functional and structural studies were performed, focusing on SmeDEF, the most relevant antibiotic- and triclosan-removing multidrug efflux pump of S. maltophilia. Expression of smeDEF is regulated by the repressor SmeT. Triclosan released SmeT from its operator and induces the expression of smeDEF, thus reducing the susceptibility of S. maltophilia to antibiotics in the presence of the biocide. The structure of SmeT bound to triclosan is described. Two molecules of triclosan were found to bind to one subunit of the SmeT homodimer. The binding of the biocide stabilizes the N terminal domain of both subunits in a conformation unable to bind DNA. To our knowledge this is the first crystal structure obtained for a transcriptional regulator bound to triclosan. This work provides the molecular basis for understanding the mechanisms allowing the induction of phenotypic resistance to antibiotics by triclosan.

Using experimental evolution to explore natural patterns between bacterial motility and resistance to bacteriophages

Resistance of bacteria to phages may be gained by alteration of surface proteins to which phages bind, a mechanism that is likely to be costly as these molecules typically have critical functions such as movement or nutrient uptake. To address this potential trade-off, we combine a systematic study of natural bacteria and phage populations with an experimental evolution approach. We compare motility, growth rate and susceptibility to local phages for 80 bacteria isolated from horse chestnut leaves and, contrary to expectation, find no negative association between resistance to phages and bacterial motility or growth rate. However, because correlational patterns (and their absence) are open to numerous interpretations, we test for any causal association between resistance to phages and bacterial motility using experimental evolution of a subset of bacteria in both the presence and absence of naturally associated phages. Again, we find no clear link between the acquisition of resistance and bacterial motility, suggesting that for these natural bacterial populations, phage-mediated selection is unlikely to shape bacterial motility, a key fitness trait for many bacteria in the phyllosphere. The agreement between the observed natural pattern and the experimental evolution results presented here demonstrates the power of this combined approach for testing evolutionary trade-offs.

Exploring the Links between Natural Products and Bacterial Assemblages in the SpongeAplysina aerophoba

The sponge Aplysina aerophoba produces a large diversity of brominated alkaloids (BAs) and hosts a complex microbial assemblage. Although BAs are located within sponge cells, the enzymes that bind halogen elements to organic compounds have been exclusively described in algae, fungi, and bacteria. Bacterial communities within A. aerophoba could therefore be involved in the biosynthesis of these compounds. This study investigates whether changes in both the concentration of BAs and the bacterial assemblages are correlated in A. aerophoba. To do so, we quantified major natural products using high-performance liquid chromatography and analyzed bacterial assemblages using denaturing gradient gel electrophoresis on the 16S rRNA gene. We identified multiple associations between bacteria and natural products, including a strong relationship between a Chloroflexi phylotype and aplysinamisin-1 and between an unidentified bacterium and aerophobin-2 and isofistularin-3. Our results suggest that these bacteria could either be involved in the production of BAs or be directly affected by them. To our knowledge, this is one of the first reports that find a significant correlation between natural products and bacterial populations in any benthic organism. Further investigating these associations will shed light on the organization and functioning of host-endobiont systems such as Aplysina aerophoba.

A fast molecular nondestructive protocol for evaluating aerobic bacterial load on fresh-cut lettuce.

Elaboration of minimally processed or fresh-cut vegetables requires a quick and reliable method for detection of bacterial contamination over the recommended limits. PCR-based methods fulfil these requirements, but amplification from DNA preparations of the food product is often hampered due to inhibiting substances. The purpose of this study was to develop a fast quantitative PCR (qPCR)-based method for aerobic bacterial enumeration in fresh-cut lettuce, using as reference the centrifugation water (CW) that comes up during processing instead of the food matrix itself. Comparisons between bacterial numbers on lettuce leaves before processing and bacterial numbers in the CW both for naturally occurring bacterial populations and for artificially inoculated lettuce were performed. On an average, 35% of the natural bacterial population and 64% of inoculated bacteria were recovered in the CW. Bacterial number in CW was proportional to initial lettuce contamination suggesting that measures on CW allow a narrow estimation of lettuce contamination. In qPCR, a 23S rDNA region was amplified from bacterial DNA present in the CW, followed by melting peak analyses and quantification. Enumeration of cell number by qPCR did not differ significantly from plate assay and might therefore replace it. The proposed protocol, which includes sample taking, DNA extraction and qPCR from the CW can be performed within less than 5 h. The resulting quantification might be used as a proxy of initial lettuce contamination, allowing direct intervention measures before fresh-cut commodity is shipped from the factory.

Mutators in cystic fibrosis chronic lung infection: Prevalence, mechanisms, and consequences for antimicrobial therapy

Hypermutable (or mutator) microorganisms are those that have an increased spontaneous mutation rate due to defects in DNA repair or error avoidance systems. Over last decade, several studies have provided strong evidence of a relevant role of mutators in the evolution of natural bacterial populations, with a special emphasis in the field of infectious diseases. Among them, chronic respiratory infection by Pseudomonas aeruginosa in cystic fibrosis (CF) patients was the first natural environment revealing a high prevalence and important role of mutators. High frequencies of mutators have also been noted for other pathogens in the CF setting, particularly Staphylococcus aureus and Haemophilus influenzae. In addition to a strong statistical link between hypermutation and antibiotic resistance (i.e., mutators are more frequently resistant against antibiotics) obtained from the analysis of collections of clinical isolates from CF patients, several in vitro and in vivo experiments have further highlighted the dramatic consequences of hypermutation, especially when acting on microorganisms that are genetically equipped to acquire efficient resistance to most antibiotics by mutations in chromosomal genes such as P. aeruginosa. Recent studies additionally showed that mutators accelerate genetic adaptation of P. aeruginosa to the airways of CF patients and that patients infected by mutator strains have a poorer lung function. Further prospective clinical studies are nevertheless needed for a deep evaluation of the impact of mutators in disease progression and outcome. Future research should particularly focus on the investigation of the optimal therapeutical strategies to prevent the emergence of mutator strains in the lungs of CF patients and to avoid multiple-antimicrobial resistance development if they have already been selected.

THE ROLE OF BACTERIALLY MEDIATED PRECIPITATION IN THE PERMINERALIZATION OF BONE

Bone fossilization is generally thought to be predominantly slow and geochemically controlled, whereas soft-tissue preservation is rapid and microbially enhanced. Microbial destruction of bone has been well researched, but potential preservational influences of microbes on bone are relatively unstudied. Building on previous work, this study examined evidence for microbially induced mineralization of bone buried in a simulated terrestrial setting using an actualistic, experimental approach designed to allow both biotic and abiotic precipitation to occur. Four trials were conducted in 2002 by burying bleached fresh bone cubes in river sand through which calcium carbonate saturated water was percolated. A “natural” trial used unmodified river sediment with natural bacterial populations. Two trials were run with bleach or sodium azide to reduce or eliminate bacterial populations. A fourth trial used washed, bleached sediment with reintroduced bacteria. After one week, the natural trial showed signs of mineral precipitation that cemented sand grains to the bone cubes. Bones from both the natural and washed trials were completely covered by adherent sediment after six weeks. After twelve weeks, cancellous bone in the nonsterile trials was permineralized while antiseptic trials and compact bone showed no indications of permineralization. Nonsterile trials also exhibited partially calcified fungal hyphae and possible lithified bacteria. This study provides additional experimental evidence that microbes are important agents of fossilization for bone as well as soft tissue. It also supports the inference that microbially induced mineral precipitation can play a significant role in bone fossilization by enhancing bone survival through early permineralization until apatite recrystallization occurs.