We determined the effects of continuous access to drinking water with a vinegar-based multi-micronutrient (VMm) supplement containing rice and fruit vinegars, vitamins, organic acids and sugars during gestation, lactation, and early adulthood in rats. Pregnant rats were provided with reverse-osmosis water or VMm water from the start of pregnancy through the time of weaning. Weaned pups consumed the same drinking water for 3 to 12 additional weeks. We examined fecal metabolite and microbial profiles, and other physiological parameters. Body weights were less in rats that drank VMm water. Thirty fecal metabolites involved in amino acid and dipeptide metabolism were significantly altered in VMm-supplemented rats. Analysis of microbial 16S rRNA showed enrichment of bacteria in the family S24-7 in VMm-supplemented rats, and one in Ruminococcaceae in controls. Our data show that a VMm-containing beverage can alter growth, and gut metabolism and microbial community. Future work to correlate these parameters is warranted.

Microbial activity has an important impact on the maintenance of cultural heritage materials, owing to the key role of microorganisms in many deterioration processes. In order to minimize such deleterious effects, there is a need to fine-tune methods that detect and characterize microorganisms. Trends in microbiology indicate that this need can be met by incorporating modern techniques. All of the methods considered in this review paper are employed in the identification, surveillance, and control of microorganisms, and they have two points in common: They are currently used in microbial ecology (only literature from 2009 to 2015 is included), and they are often applied in the cultural heritage sector. More than 75 peer-reviewed journal articles addressing three different approaches were considered: molecular, sensory and morphological, and biocontrol methods. The goal of this review is to highlight the usefulness of the traditional as well as the modern methods. The general theme in the literature cited suggests using an integrated approach.

Biodeterioration of cultural heritage artifacts due to microbial activity presents a significant challenge to conservators and museums around the World. A collection of Chinchorro mummies recovered from the Atacama Desert (the oldest artificial mummies ever found, dating back to 5050 B.C.E.) has been stored in the Universidad de Tarapacá, northern Chile. Over the past ten years, accelerated deterioration of some mummies has been documented. Blackening and exudation of some areas of their remaining skin is causing disfigurement of the mummies and poses a threat to the collection, also for mummies in situ exposed to the natural environment. This study was designed to provide a broad analysis of the skin microbiota of Chinchorro mummies and, investigate the relationship between the presence of microbes and the recent discoloration and biodegradation of the Chinchorro mummies’ skin. Microorganisms isolated from degraded Chinchorro mummy skin samples were similar, based on ribosomal RNA analysis, to bacteria found in the human skin microbiome (predominantly, Bacillus, Staphylococcus, and Methylococcus spp.) and commonly occurring fungi (predominantly, Penicillium and Aspergillus spp.). Some of these microorganisms were able to utilize collagen and/or keratin as the sole carbon source in vitro. We determined the activity of the collagenase/gelatinase enzymes produced by these microorganisms when grown on pig skin, which was used as a surrogate for human skin. The concentration of hydroxyproline, a measure of collagenous protein degradation by the microorganisms, increased with increasing relative humidity. We demonstrated that keratinolytic and collagenolytic opportunistic microorganisms were likely responsible for the recent degradation phenomenon.

Microorganisms found on graffiti and associated environments are potential candidates for biological removal of undesirable graffiti on cultural heritage structures and materials. A feasibility study involving the isolation of natural strains of microorganisms that are capable of degrading graffiti as possible candidates for use in biocleaning treatments for heritage monuments was carried out. A total of 54 different strains were obtained from various sources, recent and old graffiti, the bodywork of a car in a scrapyard and the soil beneath it, an acrylic wall painting and the interior of spray paint cans. The strains were isolated under aerobic conditions and subjected to preliminary laboratory tests to determine their potential as bioremediation agents; i.e., their ability to remove and degrade samples of paint on glass microscope slides. Only those showing such potential were further characterized by sequencing of 16S rDNA and ITS regions. Sequence results identified the isolated strains as bacteria belonging to the genera Arthrobacter, Bacillus, Gordonia, Microbacterium, Pantoea and Pseudomonas and fungi belonging to the genus Alternaria. These findings suggest that existing graffiti surfaces are a good source for putative biodegradative microbial populations, which may aid in the remediation of damaged surfaces.

Outdoor stoneworks sustain biofilm formation and are constantly at risk of deterioration by micro-organisms. In this study, the biofilm microflora of historic limestone tombstones located in a highly polluted urban environment (Cambridge, MA) and in a less polluted location (Lexington, MA) were compared using comprehensive RNA-based molecular analyses of 16S rRNA gene sequences as well as sequences of genes for different pathways of sulphur metabolism (soxB, apsA, dsrA). The metabolically active micro-organisms detected by denaturing gradient gel electrophoresis analysis of 16S rRNA fragments were predominantly represented by cyanobacteria (belonging to the family Nostocaceae and to the genus Chroococcidiopsis) in both polluted and unpolluted environments. The investigation of soxB, apsA, dsrA transcripts reflected the abundance and the diversity of sulphur-oxidizing and sulphate-reducing bacteria in the Cambridge samples in comparison with the Lexington samples. The investigation revealed that in addition to phototrophic sulphur bacteria belonging to the genera Thiocapsa, Halochromatium, Allochromatium, Thiococcus and Thermochromatium, other sulphate-oxidizing prokaryotes (e.g. the genus Thiobacillus) as well as sequences of Deltaproteobacteria from the genus Desulfovibrio occurred at the polluted urban site. The interactions between the main functional groups retrieved from the limestone tombstones were discussed.Significance and Impact of the StudyThe biofilm microflora inhabiting historic limestones are a multi-component open ecosystem sensitively reacting to all environmental factors including air pollutants. Little is known about specific target groups that are active in the biofilm and their physiological functions. For the first time, transcripts involved in important energy-yielding processes were investigated to reveal the metabolic capabilities of the microflora in response to atmospheric sulphur pollution. This work provides novel and important information about the ecology of limestone tombstone microbiota and its complex interaction with the external environment.

Foodborne diseases caused by the consumption of food contaminated with pathogenic microorganisms or their toxins have very serious economic and public health consequences. Here, we explored the effectiveness of a recently developed intervention method for inactivation of microorganisms on fresh produce, and food production surfaces. This method utilizes Engineered Water Nanostructures (EWNS) produced by electrospraying of water vapor. EWNS possess unique properties; they are 25 nm in diameter, remain airborne in indoor conditions for hours, contain Reactive Oxygen Species (ROS) and have very strong surface charge (on average 10e/structure). Here, their efficacy in inactivating representative foodborne bacteria such as Escherichia coli, Salmonella enterica, and Listeria innocua, on stainless steel surfaces and on organic tomatoes, was assessed. The inactivation was facilitated using two different exposure approaches in order to optimize the delivery of EWNS to bacteria: (1) EWNS were delivered on the surfaces by diffusion and (2) a "draw through" Electrostatic Precipitator Exposure System (EPES) was developed and characterized for EWNS delivery to surfaces. Using the diffusion approach and an EWNS concentration of 24 000 #/cm(3), the bacterial concentrations on the surfaces were reduced, depending on the bacterium and the surface type, by values ranging between 0.7 to 1.8 logs. Using the EPES approach and for an aerosol Concentration of 50 000 #/cm(3) at 90 min of exposure, results show a 1.4 log reduction for E. coli on organic tomato surfaces, as compared to the control (same conditions in regards to temperature and Relative Humidity). Furthermore, for L. innocua, the dose response relationship was demonstrated and found to be a 0.7 and 1.2 logs removal at 12 000 and 23 000 #/cm(3), respectively. The results presented here indicate that this novel, chemical-free, and environmentally friendly intervention method holds potential for development and application in the food industry, as a "green" alternative to existing disinfection methods.

From a tomb in Upper Egypt we isolated a strain of Penicillium chrysogenum that was capable of producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We present evidence that this pigment is a pyomelanin, a compound that is known to assist in the survival of some micro-organisms in adverse environments. We tested type strains of Pe . chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. Fourier transform IR analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of pyomelanin, was detected in supernatants of Pe. chrysogenum cultures growing in tyrosine medium but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium, suggesting that tyrosine induced the transcription.

This paper presents the preliminary findings from our investigation into the possible microbial origin of brown spots on the walls of Tutankhamun's tomb. GC/MS analysis of the brown spots indicated that they contained 16% (by weight) of malic acid, suggesting microbial involvement in their formation. However, no microbial structures associated with the brown spots were detected using scanning electron microscopy. Our observations indicate that the organism that created the spots is not active. We undertook an investigation of the current microbial communities on the walls in Tutankhamun's tomb and two other nearby tombs. There were no significant differences in the numbers or types of culturable microorganisms among the three tombs sampled. Using pyrosequencing, no statistically significant differences in community composition and structure were observed. Fungal communities were composed primarily of Penicillium whereas the abundant bacterial taxa were members of the Firmicutes, Actinobacteria and Bacteroidetes phyla. Penicillium chrysogenum isolated in this study produced malic acid in vitro, suggesting that they or other microorganisms may be responsible for the malic acid detected in the brown spots. Findings from this study were compared to those from previous studies, and a possible scenario for the formation of the brown spots was developed. (C) 2013 Elsevier Ltd. All rights reserved.

Cultural heritage materials are particularly susceptible to biodeterioration by fungi. Improper care and storage of artifacts contaminated with fungal material can promote the growth of these microscopic organisms and the inevitable deterioration that follows. Technology capable of detecting vegetative fungi and their reproductive structures could facilitate the struggle against fungal biodeterioration. Archivists and conservators could be notified of fungal contamination within a collection and apply pre-emptive measures, such as modification of environmental conditions, to prevent biodeterioration. The aim of this study was to improve and simplify a fluorometric assay used for the early detection of minute quantities of fungal biomass on cultural heritage materials. To this end we have successfully developed a non-fluidic assay in which fluid transfers, centrifugation steps, and much of the specialized equipment formerly needed to perform the assay are eliminated. The time required for completion of the assay was reduced to 30 min. Use of the assay was also expanded to include the early detection of viable fungal conidia from several species of fungi. These refinements will expedite implementation of this technology by archivists and conservators as they monitor and combat the fungal deterioration of cultural heritage materials.

Most, and perhaps all, multicellular organisms house symbiotic microorganisms. Recent research identified a number of ways in which symbionts are critical to the success of various insects, yet we know very little about the symbiotic associations of the major invasive species. The information we do have is largely limited to two specific types of relationships, insect vectors of plant pathogens and entomopathogens. Yet these reflect only a subset of the most prevalent types of symbioses. This presentation makes four main points: 1. Invasive insects are really invasive species complexes. 2. Symbionts can enhance the invasive potential of introduced species. 3. Understanding insect-symbiont interactions can

In a recent study, we reported a previously undescribed behavior in which a bark beetle exuded oral secretions containing bacteria that have antifungal properties, and hence defend their galleries against pervasive antagonistic Hyphomycete fungi. Actinobacteria, a group known for their antibiotic properties, were the most effective against fungi that invade the spruce beetle galleries. In the present study, we describe the isolation and identification of microorganisms from oral secretions of three bark beetles (Coleoptera: Curculionidae: Scolytinae): the spruce beetle, Dendroctonus rufipennis Kirby, the mountain pine beetle, Dendroctonus ponderosae Hopkins, and the pine engraver, Ips pini Say. Bacteria isolated from these three species span the major bacterial classes α‐, β‐, and γ‐Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, except for D. ponderosae , which yielded no α‐proteobacteria or Bacteroidetes isolates. Spruce beetles and pine engraver beetles had similar numbers of α‐proteobacteria isolates, but pine engravers yielded twice as many Bacteroidetes isolates as spruce beetles. In contrast, mountain pine beetles yielded more isolates in the β‐ and γ‐proteobacteria than spruce beetles and pine engravers. The highest percentage of Actinobacteria was obtained from spruce beetles, followed by pine engravers and mountain pine beetles. All of the fungal isolates obtained from the three beetle species were Ascomycetes. The greatest fungal diversity was obtained in spruce beetles, which had nine species, followed by pine engravers with five, and mountain pine beetles with one.

We characterized gut microbial communities in the emerald ash borer, Agrilus planipennis Fairmaire, an invasive phloem-feeding and wood-boring beetle that has caused extensive mortality to urban and forest ash trees. Analyses included both 16S rRNA gene-based and culture-based approaches. We estimated that the emerald ash borer gut harbors 44, 71, and 49 operational taxonomic units (OTUs(0.03)) in the larval, prepupal, and adult stages, respectively, and a total of 132 OTUs(0.03) when data from the three stages are pooled. The larval gut community shared all its OTUs(0.03) with either the adult or the prepupal gut community, and the adult and prepupal gut communities shared 27 OTUs(0.03). Twenty-two OTUs(0.03) were shared among the three life stages. Rarefaction analyses suggest that these gut microbial communities are close to being completely sampled at the phylum level. Culture-independent techniques yielded a higher diversity of bacteria than did culturing. Three species of bacteria inhabiting guts of emerald ash borer showed cellulolytic activity. The diverse, dynamic, and presumably multifunctional microbial community associated with emerald ash borer guts suggests that invasive insects should be viewed as multispecies complexes and that such an interpretation can improve our ability to develop more effective management approaches.

The gut bacterial community of a bark beetle, the pine engraver Ips pini (Say), was characterized using culture-dependent and culture-independent methods. Bacteria from individual guts of larvae, pupae and adults were cultured and DNA was extracted from samples of pooled larval guts. Analysis of 16S rRNA gene sequences amplified directly from the gut community suggests that the gut bacterial communities associated with I. pini are relatively simple, compared to many other systems. Six bacterial genera from four classes were detected by culturing gut bacteria from larvae, pupae and adults. Two genera, Pantoea and Stenotrophomonas (γ-Proteobacteria) were found in all life stages of I. pini, consistently in larvae and adults, and less commonly in pupae. Sequences that affiliate with the Enterobactereaceae of the γ-Proteobacteria were found in 95% of the clones sampled. The Enterobactereaceae genera, Pantoea and Erwinia, accounted for 88% of all clone sequences. These results are consistent with previous work indicating that another bark beetle, the southern pine beetle, Dendroctonus frontalis Zimmerman, also has a relatively simple gut flora, compared with wood colonizing insects such as wood borers and termites. The composition and abundance of bacteria associated with different life stages of I. pini are possibly associated with specific functions of the gut bacterial communities of larvae, pupae, and adults.

Bark beetles are known to have complex associations with a variety of microorganisms (Paine and others 1987; Ayres and others 2000; Six and Klepzig 2004). However, most of our knowledge involves fungi, particularly external species. In contrast, we know very little about their associations with bacterial gut symbionts (Bridges 1981). Similarly, work with wood colonizing insects such as termites reveals a diverse and functionally important community of gut bacteria (Hongoh and others 2005). Yet our knowledge of the gut microbiota of wood boring beetles is rudimentary. Our work is aimed at addressing these gaps, as bark and wood boring beetles include important forest pests, natural disturbance agents, and invasive species.

We report the first study of gut-associated bacteria of bark beetles using both culture-dependent and culture-independent methods. These insects are major pests of pine trees but also contribute to important ecological functions such as nutrient cycling. We found members of the alpha-and gamma-Proteobacteria and Firmicutes in larvae of the southern pine beetle, Dendroctonus frontalis Zimmermann. Sequences from three larval guts were grouped into one to three operational taxonomic units (OTUs) at 3% difference among sequences. Communities in adult southern pine beetle guts consisted solely of members of the gamma-Proteobacteria. These could be grouped into three to five OTUs at 3% difference between sequences. These gut communities have relatively low species richness, which may reflect the specialization needed to exploit a nutrient-poor food source, colonize a chemically complex habitat, and maintain consistent associations with mutualistic fungi. However, there is considerable variation in gut microbiota composition among individual insects, suggesting the need for additional studies on sources of variation and potential substitutability among species performing similar functions.