Impact of Chronic Tetracycline Exposure on Human Intestinal Microbiota in a Continuous Flow Bioreactor Model.

Studying potential dietary exposure to antimicrobial drug residues via meat and dairy products is essential to ensure human health and consumer safety. When studying how antimicrobial residues in food impact the development of antimicrobial drug resistance and disrupt normal bacteria community structure in the intestine, there are diverse methodological challenges to overcome. In this study, traditional cultures and molecular analysis techniques were used to determine the effects of tetracycline at chronic subinhibitory exposure levels on human intestinal microbiota using an in vitro continuous flow bioreactor. Six bioreactor culture vessels containing human fecal suspensions were maintained at 37 °C for 7 days. After a steady state was achieved, the suspensions were dosed with 0, 0.015, 0.15, 1.5, 15, or 150 µg/mL tetracycline, respectively. Exposure to 150 µg/mL tetracycline resulted in a decrease of total anaerobic bacteria from 1.9 × 107 ± 0.3 × 107 down to 2 × 106 ± 0.8 × 106 CFU/mL. Dose-dependent effects of tetracycline were noted for perturbations of tetB and tetD gene expression and changes in acetate and propionate concentrations. Although no-observed-adverse-effect concentrations differed, depending on the traditional cultures and the molecular analysis techniques used, this in vitro continuous flow bioreactor study contributes to the knowledge base regarding the impact of chronic exposure of tetracycline on human intestinal microbiota.

Effects of Acute and Chronic Exposure to Residual Level Erythromycin on Human Intestinal Epithelium Cell Permeability and Cytotoxicity.

Residual concentrations of erythromycin in food could result in gastrointestinal tract exposure that potentially poses a health-hazard to the consumer, affecting intestinal epithelial permeability, barrier function, microbiota composition, and antimicrobial resistance. We investigated the effects of erythromycin after acute (48 h single treatment with 0.03 µg/mL to 300 µg/mL) or chronic (repeated treatment with 0.3 µg/mL and 300 µg/mL erythromycin for five days) exposures on the permeability of human colonic epithelial cells, a model that mimics a susceptible intestinal surface devoid of commensal microbiota. Transepithelial electrical resistance (TER) measurements indicated that erythromycin above 0.3 µg/mL may compromise the epithelial barrier. Acute exposure increased cytotoxicity, while chronic exposure decreased the cytotoxicity. Quantitative PCR analysis revealed that only ICAM1 (intercellular adhesion molecule 1) was up-regulated during 0.3 µg/mL acute-exposure, while ICAM1, JAM3 (junctional adhesion molecule 3), and ITGA8 (integrin alpha 8), were over-expressed in the 300 µg/mL acute treatment group. However, during chronic exposure, no change in the mRNA expression was observed at 0.3 µg/mL, and only ICAM2 was significantly up-regulated after 300 µg/mL. ICAM1 and ICAM2 are known to be involved in the formation of extracellular matrices. These gene expression changes may be related to the immunoregulatory activity of erythromycin, or a compensatory mechanism of the epithelial cells to overcome the distress caused by erythromycin due to increased permeability.

In vitro test systems to determine tetracycline residue binding to human feces.

The use of antimicrobials, such as tetracycline, in food-producing animals may result in antimicrobial drug residues (ADR) in edible tissues from treated animals and contribute to the emergence of antibiotic resistant bacteria. The Veterinary International Conference on Harmonization (VICH) document (VICH GL36(R)/FDA-CVM Guidance for Industry#159) provides guidance on evaluating the safety of veterinary ADR in the human foods as related to effects on the human intestinal microbiome. One recognized research gap is a need for additional data and testing requirements to determine the fraction of an oral dose of ADR available to intestinal microorganisms. In the present study, we address this need by examining the binding of tetracycline to human feces using chemical and microbiological assays. High-performance liquid chromatography and liquid chromatography mass spectrometry assays showed that 25% (w/v) diluted steam sterilized feces dosed with 0.15 and 1.5 µg/ml tetracycline had binding of 58.2 ±â€¯10.8% and 56.9 ±â€¯9.1%, respectively. Tetracycline binding to fecal slurries gave similar results. Microbiological assays with two reference bacterial strains validated the results of the chemical assays. Based on data from chemical and microbiological assays methods, the fraction of dose available to microorganisms was 0.418 and 0.431 of the 0.15 and 1.5 µg/ml tetracycline treatments, respectively. This study also proposes factors to be considered when designing and conducting experiments to determine the percent of an antimicrobial agents that is available to microorganisms in the gastrointestinal tract.

An in vitro study to assess the impact of tetracycline on the human intestinal microbiome.

The human intestinal microbiome, a generally stable ecosystem, could be potentially altered by the ingestion of antimicrobial drug residues in foods derived from animals. Data and the scientific published literature on the effects of antimicrobial residues on the human intestinal microbiome are reviewed by national regulatory authorities as part of the human food safety evaluation of veterinary antimicrobial agents used in food-producing animals. In this study, we determined if tetracycline, at low residue concentrations, could impact the human intestinal microbiome structure and the resistance-gene profile, following acute and subchronic exposure. The effects of 0.15, 1.5, 15, and 150 µg/ml of tetracycline, after 24 h and 40 days of exposure, in 3% human fecal suspensions, collected from three individuals (A, B, and C) were investigated using in vitro batch cultures. Results were variable, with either no change or minor changes in total bacterial 16S rRNA gene copies after exposure of fecal samples to tetracycline, because of the inter-individual variation of human gastrointestinal tract microbiota. Bacterial community analysis using rRNA-based pyrosequencing revealed that Firmicutes and Bacteroidetes were the predominant phyla in the three fecal samples; the ratio of phylotypes varied among individuals. The evaluation of bacterial community changes at the genus level, from control to tetracycline-treated fecal samples, suggested that tetracycline under the conditions of this study could lead to slight differences in the composition of intestinal microbiota. The genus Bacteroides (of the Bacteroidetes) was consistently altered from 1.68 to 5.70% and 4.82-8.22% at tetracycline concentrations of 0.15 µg/ml or above at both time points for individual A, respectively, and increased 5.13-13.50% and 10.92-22.18% for individual B, respectively. Clostridium family XI increased 3.50-25.34% in the presence of tetracycline at 40 days for individual C. Principal Component Analysis (PCA) confirmed the pyrosequencing findings of inter-individual variability of the ratio of phylotypes and the effect of tetracycline. Among the 23 tetracycline resistance genes (TRGs) screened, four tet genes (tetO, Q, W, and X) were major TRGs in control and tetracycline-dosed fecal samples. A variable to slight increase of copy number of TRGs appeared to be related to tetracycline treatment, interindividual variability and duration of exposure. Despite, the inherent variability of the intestinal microbiota observed among or within individuals, this pilot study contributes to the knowledge base of the impact of low residue concentrations of tetracycline on the human intestinal microbiome on the potential for antimicrobial resistance.

Effects of low concentrations of erythromycin, penicillin, and virginiamycin on bacterial resistance development in vitro.

Distillers grains are co-products of the corn ethanol industry widely used in animal feed. We examined the effects of erythromycin, penicillin, and virginiamycin at low concentrations reflective of those detected in distillers grains on bacterial resistance selection. At 0.1 µg/ml erythromycin, macrolide-resistant mutants were induced in one Campylobacter coli and one Enterococcus faecium strain, while these strains plus three additional C. coli, one additional E. faecium, and one C. jejuni also developed resistance when exposed to 0.25 µg/ml erythromycin. At 0.5 µg/ml erythromycin, a total of eight strains (four Campylobacter and four Enterococcus) obtained macrolide-resistant mutants, including two strains from each genus that were not selected at lower erythromycin concentrations. For penicillin, three of five E. faecium strains but none of five Enterococcus faecalis strains consistently developed resistance at all three selection concentrations. Virginiamycin at two M1:S1 ratios did not induce resistance development in four out of five E. faecium strains; however, increased resistance was observed in the fifth one under 0.25 and 0.5 µg/ml virginiamycin selections. Although not yet tested in vivo, these findings suggest a potential risk of stimulating bacterial resistance development in the animal gut when distillers grains containing certain antibiotic residues are used in animal feed.

Effects of residual levels of tetracycline on the barrier functions of human intestinal epithelial cells.

Tetracyclines are frequently used in food-producing animals to treat, control, and prevent microbial diseases. Concerns are raised regarding the effects of residual levels of tetracycline, which may be present in the food supply, for emergence of drug-resistance and transfer of antibiotic-resistance gene in intestinal microbiota. In contrast, no information is available regarding the possible effects of residual tetracycline on the gastrointestinal epithelial layer barrier-disruption. This study investigates the outcome of tetracycline treatment on intestinal epithelial cells integrity. Intestinal epithelial cells (T84) were treated at concentrations of 0.015, 0.15, 1.5, 15 and 150 µg/ml for 48 h in an in vitro cell culture model. The permeability study revealed that 15 and 150 µg/ml of tetracycline causes barrier disruption. Whereas the altered mRNA expression of notch-3, notch-4, claudin-2, claudin-8, claudin-10, claudin-15, gap junction alpha 8 and delta 2 and integrin, alpha 3 and alpha L, which are cell-integrity-related genes starts at 1.5 µg/ml tetracycline after 48 h treatment. Translocation of GFP-labeled bacteria from apical to basal comportment provides proof of concept to intestinal barrier disruption. This study is the first to evaluate whether residual concentrations of tetracycline impact epithelial cell integrity.

An update discussion on the current assessment of the safety of veterinary antimicrobial drug residues in food with regard to their impact on the human intestinal microbiome.

The human gastrointestinal tract ecosystem consists of complex and diverse microbial communities that have now been collectively termed the intestinal microbiome. Recent scientific breakthroughs and research endeavours have increased our understanding of the important role the intestinal microbiome plays in human health and disease. The use of antimicrobial new animal drugs in food-producing animals may result in the presence of low levels of drug residues in edible foodstuffs. There is concern that antimicrobial new animal drugs in or on animal-derived food products at residue-level concentrations could disrupt the colonization barrier and/or modify the antimicrobial resistance profile of human intestinal bacteria. Therapeutic doses of antimicrobial drugs have been shown to promote shifts in the intestinal microbiome, and these disruptions promote the emergence of antimicrobial-resistant bacteria. To assess the effects of antimicrobial new animal drug residues in food on human intestinal bacteria, many national regulatory agencies and international committees follow a harmonized process, VICH GL36(R), which was issued by a trilateral organization of the European Union, the USA, and Japan called the International Cooperation on Harmonization of Technical Requirements for Veterinary Medicinal Products (VICH). The guidance describes a general approach currently used by national regulatory agencies and international committees to assess the effects of antimicrobial new animal drug residues in animal-derived food on human intestinal bacteria. The purpose of this review is to provide an overview of this current approach as part of the antimicrobial new animal drug approval process in participating countries, give insights on the microbiological endpoints used in this safety evaluation, and discuss the availability of new information. Copyright © 2016 John Wiley & Sons, Ltd.

Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-ß-α NDPSase fold differentiates NDPSases from ADPRases.

A small predatory core genome in the divergent marine Bacteriovorax marinus SJ and the terrestrial Bdellovibrio bacteriovorus.

Bacteriovorax marinus SJ is a predatory delta-proteobacterium isolated from a marine environment. The genome sequence of this strain provides an interesting contrast to that of the terrestrial predatory bacterium Bdellovibrio bacteriovorus HD100. Based on their predatory lifestyle, Bacteriovorax were originally designated as members of the genus Bdellovibrio but subsequently were re-assigned to a new genus and family based on genetic and phenotypic differences. B. marinus attaches to gram-negative bacteria, penetrates through the cell wall to form a bdelloplast, in which it replicates, as shown using microscopy. Bacteriovorax is distinct, as it shares only 30% of its gene products with its closest sequenced relatives. Remarkably, 34% of predicted genes over 500 nt in length were completely unique with no significant matches in the databases. As expected, Bacteriovorax shares several characteristic loci with the other delta-proteobacteria. A geneset shared between Bacteriovorax and Bdellovibrio that is not conserved among other delta-proteobacteria such as Myxobacteria (which destroy prey bacteria externally via lysis), or the non-predatory Desulfo-bacteria and Geobacter species was identified. These 291 gene orthologues common to both Bacteriovorax and Bdellovibrio may be the key indicators of host-interaction predatory-specific processes required for prey entry. The locus from Bdellovibrio bacteriovorus is implicated in the switch from predatory to prey/host-independent growth. Although the locus is conserved in B. marinus, the sequence has only limited similarity. The results of this study advance understanding of both the similarities and differences between Bdellovibrio and Bacteriovorax and confirm the distant relationship between the two and their separation into different families.

In vitro enrofloxacin binding in human fecal slurries.

Most antibiotic inactivation studies have been conducted through in vitro incubations of human use aminoglycosides, beta-lactams, and fluoroquinolones, usually at fecal concentrations expected with therapeutic dose regimens in humans and animals. Less is known about the inactivation of these molecules when ingested at concentrations consistent with residue levels present in animal-derived foods from antibiotic treated animals. In this investigation, we used the fluoroquinolone, enrofloxacin which is specifically marketed for veterinary medicine as test compound. Fecal suspensions at 10%, 25%, and 50% (w/v) were subjected to physicochemical and molecular characterization and used in the drug binding studies. The fecal binding of enrofloxacin added at concentrations of 0.06, 0.1, 1, 5, 15, 50, and 150 mg/L was determined in various fecal slurry suspensions using analytical chemistry and microbiological assay methods. There was consistent correlation between both assay methods. By the analytical chemistry assay, the 10%, 25% and 50% diluted autoclaved fecal samples dosed with enrofloxacin showed binding of 50±4.6%, 54±6.5% and 56±6.8% of the enrofloxacin, respectively. Binding of enrofloxacin to fecal contents occurred rapidly within 10 min and remained constant over the incubation period. Denaturing gradient gel electrophoreses and pyrosequencing analysis showed varied profiles of the bacterial composition of the human intestinal microbiota for fecal samples from different individuals. This study provided information on methodological questions that have concerned regulatory authorities on in vitro testing to determine if concentrations of veterinary antimicrobial agent residues entering the human colon remain microbiologically active.

Structure and biological function of the RNA pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus.

Until recently, the mechanism of mRNA decay in bacteria was thought to be different from that of eukaryotes. This paradigm changed with the discovery that RppH (ORF176/NudH/YgdP), an Escherichia coli enzyme that belongs to the Nudix superfamily, is an RNA pyrophosphohydrolase that initiates mRNA decay by cleaving pyrophosphate from the 5'-triphosphate. Here we report the 1.9 Angstroms resolution structure of the Nudix hydrolase BdRppH from Bdellovibrio bacteriovorus, a bacterium that feeds on other Gram-negative bacteria. Based on the structure of the enzyme alone and in complex with GTP-Mg2+, we propose a mode of RNA binding similar to that of the nuclear decapping enzyme from Xenopus laevis, X29. In additional experiments, we show that BdRppH can indeed function in vitro and in vivo as an RNA pyrophosphohydrolase. These findings set the basis for the identification of possible decapping enzymes in other bacteria.

Bacterial intein-like domains of predatory bacteria: a new domain type characterized in Bdellovibrio bacteriovorus.

We report a new family of bacterial intein-like domains (BILs) identified in ten proteins of four diverse predatory bacteria. BILs belong to the HINT (Hedgehog/Intein) superfamily of domains that post-translationally self-process their protein molecules by protein splicing and self-cleavage. The new, C-type, BILs appear with other domains, including putative predator-specific domain 1 (PPS-1), a new domain typically appearing immediately upstream of C-type BILs. The Bd2400 protein of the obligate predator Bdellovibrio bacteriovorus includes a C-type BIL and a PPS-1 domains at its C-terminal part, and a signal peptide and two polycystic kidney disease domains at its N-terminal part. We demonstrate the in vivo transcription, translation, secretion, and processing of the B. bacteriovorus protein, and the in vitro autocatalytic N-terminal cleavage activity of its C-type BIL. Interestingly, whereas the Bd2400 gene is constitutively expressed, its protein product is differentially processed throughout the dimorphic life cycle of the B. bacteriovorus predator. The modular structure of the protein, its localization, and complex processing suggest that it may be involved in the interaction between the predator and its prey.

Identification of Bdellovibrio bacteriovorus HD100 Bd0714 as a Nudix dGTPase.

Bdellovibrio bacteriovorus bacteria are predatory organisms that attack other gram-negative bacteria. Here, we report that Bd0714 is a Nudix dGTPase from B. bacteriovorus HD100 with a substrate specificity similar to that of Escherichia coli MutT and complements an E. coli mutT-deficient strain. We observed different transcription levels of the gene throughout the predator life cycle.

Development and evaluation of a quantitative real-time PCR assay for the detection of saltwater Bacteriovorax.

Bdellovibrio-and-like-organisms (BALOs) are small, Gram-negative predatory bacteria with the ability to prey on a wide variety of Gram-negative bacteria, and which may have a significant ecological role. Detection and quantification of BALOs by culture-dependent methods are complicated, as their reproduction is dependent upon the use of appropriate prey. For this reason, a sensitive and specific molecular detection method was developed. This paper describes a SYBR Green-based real-time PCR (quantitative PCR) assay that combines the use of a specific 16S rDNA primer with a universal primer for quantitative detection of halophilic Bacteriovorax. 16S rDNA sequences from 174 BALO strains, including both halophilic and freshwater, were aligned and a consensus region was identified that is unique to the halophilic Bacteriovorax strains. A specific primer was designed and analysed for specificity. The PCR conditions were optimized to obtain high specificity and sensitivity. The specificity was evaluated by testing a series of halophilic Bacteriovorax samples and prey specimens, including both pure cultures and environmental saltwater samples. A linear and reproducible standard curve was obtained over a range of 10(1)-10(6) gene copies and the detection limit was determined to be 10 copies of 16S rRNA gene per reaction. The results presented in this study validate the procedure as a rapid, sensitive and accurate method for the detection and quantification of halophilic Bacteriovorax in environmental saltwater samples. It is anticipated that this culture-independent method will facilitate future investigations of the distribution and population dynamics of these interesting predatory bacteria, leading to a better understanding of their ecological role.

Phylogenetic relationships amongst the saltwater members of the genus Bacteriovorax using rpoB sequences and reclassification of Bacteriovorax stolpii as Bacteriolyticum stolpii gen. nov., comb. nov.

Members of the saltwater genus Bacteriovorax, formerly known as the marine Bdellovibrio, are obligate predatory bacteria that prey selectively on other Gram-negative bacteria. Previous phylogenetic analysis based on the 16S rRNA genes of saltwater Bacteriovorax isolates from environmental samples revealed 11 distinct phylogenetic clusters based on > or =96.5 % gene sequence similarity. In other micro-organisms, the gene coding for the beta-subunit of RNA polymerase (rpoB) has been shown to be more discriminating than 16S rRNA genes. In this study, rpoB sequences from Bacteriovorax isolates were analysed to determine whether the results would be consistent with those based on 16S rRNA gene sequences. A 1242 bp region of the rpoB gene from 74 saltwater Bacteriovorax strains and two freshwater isolates, Bacteriovorax stolpii Uki2T and Peredibacter starrii A3.12T, was amplified by PCR and analysed. The sequences were aligned and phylogenetic trees were constructed using a neighbour-joining algorithm. The resulting tree showed that the rpoB sequences produced smaller subdivisions of isolates, but were nevertheless consistent with the clusters determined using 16S rRNA gene sequences. Thus, the highly conserved 16S rRNA gene sequences provided good phylogenetic information and the rpoB gene sequences permitted greater differentiation in order to further subdivide phylogenetically distinct groups within the genus Bacteriovorax. Also, on the basis of the extensive diversity and large distance between the saltwater members of the genus Bacteriovorax and the freshwater/soil Bacteriovorax, a reclassification of Bacteriovorax stolpii as Bacteriolyticum stolpii gen. nov., comb. nov. is proposed. A new family, Peredibacteraceae fam. nov., is also described.

Global survey of diversity among environmental saltwater Bacteriovoracaceae.

Halophilic Bacteriovorax (Bx), formerly known as the marine Bdellovibrio, are Gram-negative, predatory bacteria found in saltwater systems. To assess their genetic diversity and geographical occurrence, the small subunit rRNA (ssu-rRNA) gene sequences were analysed from 111 marine, salt lake and estuarine isolates recovered from 27 locations around the world. Phylogenetic analysis of these isolates using Geobacter as the outgroup revealed eight distinct ribotype clusters each with at least two isolates. Each cluster was composed of isolates with >or= 96.5% similarity in ssu-rRNA sequences. Three single isolate outliers were observed. Many of the Bx ribotypes were widely dispersed among different types of ecosystems (e.g. cluster III was recovered from the Great Salt Lake, the Atlantic Ocean, Pacific Ocean, Chesapeake Bay and gills of aquarium fish). However, cluster V was only recovered from a single ecosystem, estuaries. Cluster V was originally detected in the Chesapeake Bay and subsequently in the Pamlico Sound/Neuse River system. Principal coordinate analysis revealed that the sequences of the isolates from different environments were distinct from each other. The results of this study reveal the saltwater Bx to be phylogenetically and environmentally more diverse than was previously known.

Development of a novel genetic system to create markerless deletion mutants of Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is a species of unique obligate predatory bacteria that utilize gram-negative bacteria as prey. Their life cycle alternates between a motile extracellular phase and a growth phase within the prey cell periplasm. The mechanism of prey cell invasion and the genetic networks and regulation during the life cycle have not been elucidated. The obligate predatory nature of the B. bacteriovorus life cycle suggests the use of this bacterium in potential applications involving pathogen control but adds complexity to the development of practical genetic systems that can be used to determine gene function. This work reports the development of a genetic technique for allelic exchange or gene inactivation by construction of in-frame markerless deletion mutants including the use of a counterselectable marker in B. bacteriovorus. A suicide plasmid carrying the sacB gene for counterselection was used to inactivate the strB gene in B. bacteriovorus HD100 by an in-frame deletion. Despite the inactivation of the strB gene, B. bacteriovorus was found to retain resistance to high concentrations of streptomycin. The stability of a plasmid for use in complementation experiments was also investigated, and it was determined that pMMB206 replicates autonomously in B. bacteriovorus. Development of this practical genetic system now facilitates the study of B. bacteriovorus at the molecular level and will aid in understanding the regulatory networks and gene function in this fascinating predatory bacterium.

Reclassification of salt-water Bdellovibrio sp. as Bacteriovorax marinus sp. nov. and Bacteriovorax litoralis sp. nov.

Bdellovibrios are unique, predatory bacteria with an intraperiplasmic growth and multiplication phase within their prey, which consists of many Gram-negative bacteria. Until recently, all bacteria that exhibited these traits were included in the genus Bdellovibrio. However, analysis of 16S rDNA sequences and other studies have demonstrated substantial genotypic, phenotypic and ecotypic diversity among the organisms in this genus (Baer et al., 2000; Snyder et al., 2002). This has resulted in reclassification of Bdellovibrio stolpii and Bdellovibrio starrii into the newly constructed genus Bacteriovorax (Baer et al., 2000). In this study, examination of marine isolates of Bdellovibrio (designated SJT, AQ and JS5T) has revealed them to be related more closely to the newly designated genus Bacteriovorax. Phylogenetic analysis of 16S rRNA gene sequences revealed that marine isolates SJT, AQ and JS5T clustered in a separate clade from Bdellovibrio bacteriovorus 100T as part of the clade that contains Bacteriovorax spp., indicating a much closer taxonomic relationship to the latter. DNA-DNA hybridization experiments also demonstrated <5 % similarity between Bdellovibrio bacteriovorus 100T and the marine isolates. Distinct differences between the salt-water group and Bdellovibrio spp. were also observed by determination of DNA G+C content, salinity growth testing and antibiotic sensitivity analysis. On the basis of the results from the studies described above, it is proposed that marine isolates SJT (=ATCC BAA-682T=DSM 15412T) and JS5T (=ATCC BAA-684T=DSM 15409T) should be classified within the genus Bacteriovorax as the type strains of Bacteriovorax marinus sp. nov. and Bacteriovorax litoralis sp. nov., respectively.

Discrepancies in bacterial recovery from dental unit water samples on R2A medium and a commercial sampling device.

Monitoring the number of bacterial colony-forming units is an important step in assuring compliance with the recommendation that water from dental units contain <200 CFU mL(-1). Media that have been used for this purpose include R2A, a standard plate counting medium for water samples, and the Millipore HPC Sampler device, designed to facilitate sampling in dental offices. Discrepancies between the two media have been observed. This study tested the hypothesis that differences in counts on the two media were due to the failure of some bacteria to grow on the HPC sampler or to grow at less efficiency than on R2A. Of four different bacterial colony phenotypes tested in three independent experimental trials, one phenotype did not grow on the HPC device, and another grew inconsistently and at lower efficiency. These results confirmed the hypothesis. From these findings, users of the HPC sampler should be aware that microbial undercounts may occur.

Predation pattern and phylogenetic analysis of Bdellovibrionaceae from the Great Salt Lake, Utah.

The Bdellovibrionaceae are predatory, intraperiplasmic bacteria that prey upon a variety of Gram-negative bacteria. The prey susceptibility pattern is frequently used to characterize new isolates. The objective in this study was to isolate and characterize predators from the Great Salt Lake (GSL) by prey susceptibility testing. To recover the predators, water samples were inoculated into an enrichment medium with Vibrio parahaemolyticus as prey. After several days of incubation, the predators were isolated, pure DNA was extracted, and partial 16S rDNA gene was sequenced. Water samples were also plated for isolation of heterotrophic bacteria. The susceptibility of bacterial isolates from the lake and other sources to each predator isolate was determined. The results revealed that there are predators in the GSL, and they preferentially prey on bacteria from the lake. This is the first report of the isolation of Bdellovibrionaceae from GSL and the predators showing preferences for bacteria from the same habitat.