Detection and Identification of tdh- and trh-Positive Vibrio parahaemolyticus Strains from Four Species of Cultured Bivalve Molluscs on the Spanish Mediterranean Coast

Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.Gastroenteritis caused by Vibrio parahaemolyticus has been reported worldwide, though only sporadic cases have been reported in Europe (7, 14). The bacterium can be naturally present in seafood, but pathogenic isolates capable of inducing gastroenteritis in humans are rare in environmental samples (2 to 3%) (15) and are often not detected (10, 19, 20).The virulence of V. parahaemolyticus is based on the presence of a thermostable direct hemolysin (tdh) and/or the thermostable direct hemolysin-related gene (trh) (1, 5). Both are associated with gastrointestinal illnesses (2, 9).Spain is not only the second-largest producer in the world of live bivalve molluscs but also one of the largest consumers of bivalve molluscs, and Catalonia is the second-most important bivalve producer of the Spanish Autonomous Regions. Currently, the cultivation of bivalves in this area is concentrated in the delta region of the Ebro River. The risk of potentially pathogenic Vibrio spp. in products placed on the market is not assessed by existing legislative indices of food safety in the European Union, which emphasizes the need for a better knowledge of the prevalence of diarrheal vibrios in seafood products. The aim of this study was to investigate the distribution and pathogenic potential of V. parahaemolyticus in bivalve species exploited in the bays of the Ebro delta.Thirty animals of each species of Mytilus galloprovincialis, Crassostrea gigas, Ruditapes decussatus, and Ruditapes philippinarum were collected. They were sampled from six sites of the culture area, three in each bay of the Ebro River delta, at the beginning (40°37′112"N, 0°37′092"E [Alfacs]; 40°46′723"N, 0°43′943"E [Fangar]), middle (40°37′125"N, 0°38′570"E [Alfacs]; 40°46′666"N, 0°45′855"E [Fangar]), and end (40°37′309"N, 0°39′934"E [Alfacs]; 40°46′338"N, 0°44′941"E [Fangar]) of the culture polygon. Clams were sampled from only one site per bay as follows: in the Alfacs Bay from a natural bed of R. decussatus (40°37′44"N, 0°38′0"E) and in the Fangar Bay from an aquaculture bed of R. philippinarum (40°47′3"N, 0°43′8"E). In total, 367 samples were analyzed in 2006 (180 oysters, 127 mussels, 30 carpet shell clams, and 30 Manila clams) and 417 samples were analyzed in 2008 (178 oysters, 179 mussels, 30 carpet shell clams, and 30 Manila clams).All animals were individually processed and homogenized, and 1 ml of the homogenate was inoculated into 9 ml of alkaline peptone water (Scharlau, Spain). Following a 6-h incubation at 37°C, one loopful of the contents of each tube of alkaline peptone water was streaked onto CHROMagar vibrio plates (CHROMagar, France) and incubated for 18 h at 37°C. Mauve-purple colonies were purified, and each purified isolate was cryopreserved at −80°C (135 isolates in 2006 and 96 in 2008). From the initial homogenate portion, 100 μl was inoculated onto marine agar (Scharlau, Spain) and onto thiosulfate citrate-bile salts-sucrose agar (Scharlau, Spain) for total heterotrophic marine bacteria counts and total vibrio counts, respectively (Table ​(Table11).

TABLE 1.

Vibrio parahaemolyticus isolates, serotypes, and origins and total number of vibrios/heterotrophic bacteria contained in the bivalve^a

Transfer of a Phage T4 Gene into Enterobacteriaceae,Determined at the Single-Cell Level

The transfer range of phage genes was investigated at the single-cell level by using an in situ DNA amplification technique. After absorption of phages, a phage T4 gene was maintained in the genomes of non-plaque-forming bacteria at frequencies of 10⁻² gene copies per cell. The gene transfer decreased the mutation frequencies in nonhost recipients.Recently, whole-genome analyses have revealed that many bacterial genomes contain foreign genes, especially phage genes (9). The phage genes in bacterial genomes include genes for virulence or fitness factors such as extracellular toxins, superantigens, lipopolysaccharide-modifying enzymes, and proteins conferring serum resistance, etc. (1). These findings suggest that the horizontal transfer of phage genes has contributed significantly to the acquisition of new genetic traits and to the genetic diversity of bacteria (1, 9, 10). To truly appreciate the mechanisms behind phage-associated evolution, it is important to understand the frequency and range of transfer of phage genes.Most phage genomes consist of many genes derived from different origins (5, 8). Some genes are similar to those of other phages with phylogenetically different hosts or are found in the genomes of bacteria that are not the phage hosts. The mosaic nature of phage genomes has been known for some time, and a body of molecular genetic studies of phages to explain the mechanisms that drive this feature have been attempted previously (1, 5). More importantly, the horizontal transfer of phage genes has emerged as a major factor in the evolution of the phage genome. Since recombination between phage and phage/prophage can occur when these elements coexist in the same cell, coinfection with multiple phage species may result in the production of hybrid phage genomes (5). The pathways by which phages exchange genetic material vary dramatically in concert with host ranges. However, conventional plaque assays have shown that the host ranges of the phages studied are narrow. We hypothesized that phage genes can be transferred to more diverse species than previously thought.In order to accurately quantify DNA movement, gene targeting that does not require cultivation or gene expression is necessary (7). In situ DNA amplification methods allow the visualization of specific DNA sequences inside bacterial cells. In this study, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) to examine the possible range and frequency of the transfer of phage genes. CPRINS uses one primer and results in linear amplification of the target DNA inside cells, and multiply labeled fluorescent probe sets are applied for detection of the amplicons to improve the specificity and sensitivity of CPRINS (3). Previously, CPRINS-FISH did clarify the movement of DNA of a specific gene among Escherichia coli cells at the single-cell level (4).Enterobacterial phages P1 and T4 infect E. coli and have been well studied. P1 can exist as circular DNA within the bacterial cell as if it were a plasmid. Phage T4 is capable of undergoing only a lytic life cycle and not the lysogenic life cycle. Conventional methods using plaque assays have shown that the host of P1 and T4 is E. coli, but orthologous phage genes have been found in bacteria other than E. coli (6, 8). In the present study, strains of Enterobacteriaceae were allowed to grow on agar medium after the phage was adsorbed, and the maintenance of the transferred phage gene in the bacterial genomes was examined at the community level by quantitative real-time PCR and at the single-cell level by CPRINS-FISH.The following bacterial strains were used for maintenance experiments: Citrobacter freundii IFO 12681, Enterobacter aerogenes BM 2688, E. coli NBRC 12713, a Proteus mirabilis clinical isolate, Salmonella enterica serovar Enteritidis IID 640, and Yersinia enterocolitica IID 981. The bacterial strains were grown in Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl; Nacalai Tesque Inc., Kyoto, Japan) at 37°C overnight.Stationary-phase cultures of 500 μl were incubated with 500 μl of SM buffer (50 mmol liter⁻¹ Tris-HCl [pH 7.5], 100 mmol liter⁻¹ NaCl, 8 mmol liter⁻¹ MgSO₄, 0.01% gelatin) containing the phage P1kc NBRC 20008 (2) or T4GT7 (11) at 37°C for 10 min at a multiplicity of infection of 1:1 (ratio of PFU of the phage to CFU of the recipient bacterium). The concentration of bacterial cells was adjusted to 10⁹ cells ml⁻¹. After 10 min of incubation, the diluted cell suspension (10⁵ cells) was filtered through a polycarbonate filter (Advantec, Tokyo, Japan) with a pore size of 0.2 μm and a diameter of 25 mm. Cells trapped on the filter were cultured on LB agar medium at 37°C for 24 h. The filter was transferred into a microtube, and cells on the filter were suspended in 1 ml of sterile deionized water. The numbers of cells in the suspension and cells remaining on the filter were determined by using an epifluorescence microscope (see below) after staining of the samples with 1 μg ml⁻¹ of 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Japan, Tokyo). The level of recovery of cells from the filter into sterile deionized water was about 99%. The cultured cells were subjected to real-time PCR and CPRINS-FISH.For real-time PCR, bacterial DNA was extracted using a QIAamp DNA isolation kit (Qiagen, Tokyo, Japan). The cell suspension was mixed with 10 mg ml⁻¹ of lysozyme solution and incubated at 37°C for 1 h. DNA extraction was then performed according to the kit manufacturer''s instructions. Table ​Table11 lists the oligonucleotide primers for PCR and CPRINS and the polynucleotide probes used in the present study. Tail fiber genes from phages P1kc and T4GT7 were quantified by real-time PCR with a LightCycler system (Roche Diagnostics, Tokyo, Japan). LightCycler FastStart DNA master SYBR green I (Roche Diagnostics) was used with 5 mmol liter⁻¹ Mg²⁺ and 0.5 μmol liter⁻¹ (each) primers targeting the tail fiber genes of P1kc (P1-tail931f and P1-tail1148r) and T4GT7 (T4-tail2770f and T4-tail2983r). After a hot start for 10 min at 95°C, 40 cycles of PCR were run with denaturation at 94°C for 15 s, annealing at 60°C for 10 s, extension at 72°C for 10 s, and fluorescence detection at 83°C for 5 s. The known amounts of PCR products from the phage DNA (10¹ to 10⁷ copies per reaction) were used for the standard curves to quantify the target DNA. To confirm the specificity of the reaction after real-time PCR, the PCR mixture was collected in a glass capillary and subjected to agarose gel electrophoresis in addition to a melting-curve analysis with the LightCycler system. The maintenance frequencies determined by real-time PCR were recorded as the copy number of the phage tail fiber gene per bacterial genome detected by staining with PicoGreen (Invitrogen, Tokyo, Japan) after cultivation of cells on LB agar medium for 24 h as described above. The frequencies were determined in triplicate for each sample. The increase in the phage gene copy number was determined by comparing the copy numbers in cells on the filter before and after cultivation. The phage gene copy number in cells on the filter was determined by the following formula: (total number of cells determined by DAPI staining) × (phage tail fiber gene copy number determined by real-time PCR)/(bacterial genome copy number determined by PicoGreen staining).

TABLE 1.

Probes and primers designed in this study

Antimicrobial Activity of Simulated Solar Disinfection against Bacterial,Fungal, and Protozoan Pathogens and Its Enhancement by Riboflavin

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m⁻²) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log₁₀ after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log₁₀ after 6 h) was obtained only in the presence of riboflavin and 250 W m⁻² irradiance.Solar disinfection (SODIS) is an established and proven technique for the generation of safer drinking water (11). Water is collected into transparent plastic polyethylene terephthalate (PET) bottles and placed in direct sunlight for 6 to 8 h prior to consumption (14). The application of SODIS has been shown to be a simple and cost-effective method for reducing the incidence of gastrointestinal infection in communities where potable water is not available (2-4). Under laboratory conditions using simulated sunlight, SODIS has been shown to inactivate pathogenic bacteria, fungi, viruses, and protozoa (6, 12, 15). Although SODIS is not fully understood, it is believed to achieve microbial killing through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating (21).The combination of UVA radiation and riboflavin (vitamin B₂) has recently been reported to have therapeutic application in the treatment of bacterial and fungal ocular pathogens (13, 17) and has also been proposed as a method for decontaminating donor blood products prior to transfusion (1). In the present study, we report that the addition of riboflavin significantly enhances the disinfectant efficacy of simulated SODIS against bacterial, fungal, and protozoan pathogens.Chemicals and media were obtained from Sigma (Dorset, United Kingdom), Oxoid (Basingstoke, United Kingdom), and BD (Oxford, United Kingdom). Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), and Fusarium solani (ATCC 36031) were obtained from ATCC (through LGC Standards, United Kingdom). Escherichia coli (JM101) was obtained in house, and the Legionella pneumophila strain used was a recent environmental isolate.B. subtilis spores were produced from culture on a previously published defined sporulation medium (19). L. pneumophila was grown on buffered charcoal-yeast extract agar (5). All other bacteria were cultured on tryptone soy agar, and C. albicans was cultured on Sabouraud dextrose agar as described previously (9). Fusarium solani was cultured on potato dextrose agar, and conidia were prepared as reported previously (7). Acanthamoeba polyphaga (Ros) was isolated from an unpublished keratitis case at Moorfields Eye Hospital, London, United Kingdom, in 1991. Trophozoites were maintained and cysts prepared as described previously (8, 18).Assays were conducted in transparent 12-well tissue culture microtiter plates with UV-transparent lids (Helena Biosciences, United Kingdom). Test organisms (1 × 10⁶/ml) were suspended in 3 ml of one-quarter-strength Ringer''s solution or natural freshwater (as pretreated water from a reservoir in United Kingdom) with or without riboflavin (250 μM). The plates were exposed to simulated sunlight at an optical output irradiance of 150 watts per square meter (W m⁻²) delivered from an HPR125 W quartz mercury arc lamp (Philips, Guildford, United Kingdom). Optical irradiances were measured using a calibrated broadband optical power meter (Melles Griot, Netherlands). Test plates were maintained at 30°C by partial submersion in a water bath.At timed intervals for bacteria and fungi, the aliquots were plated out by using a WASP spiral plater and colonies subsequently counted by using a ProtoCOL automated colony counter (Don Whitley, West Yorkshire, United Kingdom). Acanthamoeba trophozoite and cyst viabilities were determined as described previously (6). Statistical analysis was performed using a one-way analysis of variance (ANOVA) of data from triplicate experiments via the InStat statistical software package (GraphPad, La Jolla, CA).The efficacies of simulated sunlight at an optical output irradiance of 150 W m⁻² alone (SODIS) and in the presence of 250 μM riboflavin (SODIS-R) against the test organisms are shown in Table ​Table1.1. With the exception of B. subtilis spores and A. polyphaga cysts, SODIS-R resulted in a significant increase in microbial killing compared to SODIS alone (P < 0.001). In most instances, SODIS-R achieved total inactivation by 2 h, compared to 6 h for SODIS alone (Table ​(Table1).1). For F. solani, C. albicans, ands A. polyphaga trophozoites, only SODIS-R achieved a complete organism kill after 4 to 6 h (P < 0.001). All control experiments in which the experiments were protected from the light source showed no reduction in organism viability over the time course (results not shown).

TABLE 1.

Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)

Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages

Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.Classically, transducing phages use the pac site-headful system for DNA packaging. Packaging is initiated on concatemeric post-replicative DNA by terminase cleavage at the sequence-specific pac site, a genome slightly longer than unit length is packaged, and packaging is completed by non-sequence-specific cleavage (reviewed in Rao and Feiss, 2008). Generalized transduction results from the initiation of packaging at pac site homologs in host chromosomal or plasmid DNA, and typically represents ∼1% of the total number of phage particles. In the alternative cos site mechanism packaging is also initiated on concatemeric post-replicative DNA by terminase cleavage at a sequence-specific (cos) site. Here, however, packaging is completed by terminase cleavage at the next cos site, generating a precise monomer with the cohesive termini used for subsequent circularization (Rao and Feiss, 2008). Although cos site homologs may exist in host DNA, it is exceedingly rare that two such sites would be appropriately spaced. Consequently, cos phages, of which lambda is the prototype, do not engage in generalized transduction. For this reason, cos-site phages have been preferred for possible phage therapy, since they would not introduce adventitious host DNA into target organisms.The Staphylococcus aureus pathogenicity islands (SaPIs) are the best-characterized members of the phage-inducible chromosomal island family of mobile genetic elements (MGEs; Novick et al., 2010). SaPIs are ∼15 kb mobile elements that encode virulence factors and are parasitic on specific temperate (helper) phages. Helper phage proteins are required to lift their repression (Tormo-Más et al., 2010, 2013), thereby initiating their excision, circularization and replication. Phage-induced lysis releases vast numbers of infectious SaPI particles, resulting in high frequencies of transfer. Most SaPI helper phages identified to date are pac phages, and many well-studied SaPIs are packaged by the headful mechanism (Ruzin et al., 2001; Ubeda et al., 2007). Recently, we have reported that some SaPIs, of which the prototype is SaPIbov5 (Viana et al., 2010), carry phage cos sequences in their genomes, and can be efficiently packaged and transferred by cos phages to S. aureus strains at high frequencies (Quiles-Puchalt et al., 2014). Here we show that this transfer extends to non-aureus staphylococci and to Listeria monocytogenes.Since the pac phages transfer SaPIs to non-aureus staphylococci and to the Gram-positive pathogen Listeria monocytogenes (Maiques et al., 2007; Chen and Novick, 2009), we reasoned that cos phages might also be capable of intra- and intergeneric transfer. We tested this with SaPIbov5, into which we had previously inserted a tetracycline resistance (tetM) marker to enable selection, and with lysogens of two helper cos phages, φ12 and φSLT, carrying SaPIbov5 (strains JP11010 and JP11194, respectively; Supplementary Table 1). The prophages in these strains were induced with mitomycin C, and the resulting lysates were adjusted to 1 μg ml⁻¹ DNase I and RNase A, filter sterilized (0.2 μm pore), and tested for SaPI transfer with tetracycline selection, as previously described (Ubeda et al., 2008). To test for trans-specific or trans-generic transduction, coagulase-negative staphylococci species and L. monocytogenes strains were used as recipients for SaPIbov5 transfer, respectively, as previously described (Maiques et al., 2007; Chen and Novick, 2009). As shown in Figure 1 and Supplementary Table 2). In contrast, deletion of the SaPIbov5 cos site (strains JP11229 and JP11230) did not affect SaPI replication (Supplementary Figure 1), but completely eliminated SaPIbov5 transfer (Supplementary Table 2). The TerS protein is essential for φ12 and SaPIbov5 DNA packaging, but not for phage-mediated lysis (Quiles-Puchalt et al., 2014). As expected, this mutation abolished SaPIbov5 transfer (Open in a separate windowFigure 1(a) Map of SaPIbov5. Arrows represent the localization and orientation of ORFs greater than 50 amino acids in length. Rectangles represent the position of the ori (in purple) or cos (in red) sites. Positions of different primers described in the text are shown. (b) Amplimers generated for detection of SaPIbov5 in the different recipient strains. Supplementary Table 2 lists the sequence of the different primers used. The element was detected in S. epidermidis JP829 (Se-1), S. epidermidis JP830 (Se-2), L. monocytogenes SK1351 (Lm-1), L. monocytogenes EGDe (Lm-2), S. xylosus C2a (Sx) and S. aureus JP4226 (Sa).

Table 1

Intra- and intergeneric SaPIbov5 transfer^a

Impact of Viral Dose and Major Histocompatibility Complex Class IB Haplotype on Viral Outcome in Mauritian Cynomolgus Monkeys Vaccinated with Tat upon Challenge with Simian/Human Immunodeficiency Virus SHIV89.6P

The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated (n = 67) or not vaccinated (n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6P_cy243. In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID₅₀]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load (P = 0.0280) and CD4 loss (P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID₅₀ (P < 0.0001), and contained acute CD4 loss at 15 MID₅₀ (P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility (P = 0.0199) and resistance (P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion (P = 0.0391) during chronic infection, independently of the challenge dose or haplotype.Advances in typing of the major histocompatibility complex (MHC) of Mauritian cynomolgus macaques (14, 20, 26) have provided the opportunity to address the influence of host factors on vaccine studies (13). Retrospective analysis of 22 macaques vaccinated with Tat or a Tat-expressing adenoviral vector revealed that monkeys with the H6 or H3 MHC class IB haplotype were overrepresented among aviremic or controller animals, whereas macaques with the H2 or H5 haplotype clustered in the noncontrollers (12). More recently, the H6 haplotype was reported to correlate with control of chronic infection with simian immunodeficiency virus (SIV) mac251, regardless of vaccination (18).Here, we performed a retrospective analysis of 112 Mauritian cynomolgus macaques, which included the 22 animals studied previously (12), to evaluate the impact of the challenge dose and class IB haplotype on the acquisition and severity of simian/human immunodeficiency virus (SHIV) 89.6P_cy243 infection in 45 control monkeys and 67 monkeys vaccinated with Tat from different protocols (Table ​(Table11).

TABLE 1.

Summary of treatment, challenge dose, and outcome of infection in cynomolgus monkeys

Widespread Distribution of Poribacteria in Demospongiae

Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.Recently, a novel bacterial phylum, termed “Poribacteria,” was discovered, and members of this phylum have been found exclusively in sponges (2). Phylogenetic analyses of 16S rRNA genes indicated that poribacteria are evolutionarily deeply branching organisms and related to a superphylum composed of Planctomycetes, Verrucomicrobia, and Chlamydia (11). Poribacterial 16S rRNA genes contain 13 of 15 planctomycete signature nucleotides, but a level of sequence divergence of more than 25% compared to any other bacterial phylum, including the Planctomycetes, justifies the status of this taxon as an independent phylum. A consistent treeing pattern is difficult to resolve in comparative phylogenetic sequence analyses, making the poribacteria an unusual line of phylogenetic descent. In addition to their divergent status as a separate phylum on the basis of the 16S rRNA sequence, poribacteria are also divergent because they may have a compartmentalized cell structure, a cell plan they share only with members of the phyla Planctomycetes and Verrucomicrobia (2). They are also of interest for understanding the potential contribution of obligate sponge-associated bacteria to the sponges harboring them and as an example of a yet-to-be-cultured group of bacteria associated with invertebrate tissue apparently exclusively but for unknown reasons. This study aimed to further explore the presence and diversity of poribacteria in different marine demosponge genera using samples from around the world.The Mediterranean sponges were collected by scuba divers offshore at Banyuls sur Mer, France (42°29′N, 03°08′E). The Caribbean sponges were collected offshore at Little San Salvador Island, Bahamas (24°32′N, 75°55′W). The eastern Pacific sponge Aplysina fistularis was collected offshore at San Diego, CA (32°51′N, 117°15′W). The western Pacific sponge Theonella swinhoei was collected offshore at Palau (07°23′N, 134°38′E). All non-Great Barrier Reef (non-GBR) sponges were collected between May and July 2000, and once individual sponge specimens were brought to the surface, they were frozen in liquid nitrogen on board ship and stored at −80°C until microbiological processing (9). The GBR marine sponges were collected off Heron Island Research Station (23°27′S, 151°5′E) in April 2002 (5). Pseudoceratina clavata was collected by scuba divers at a depth of 14 m, and Rhabdastrella globostellata was collected at a depth of ca. 0.5 m after a reef walk consisting of a few hundred meters. The samples were immediately placed in plastic bags and brought to Heron Island Research Station, where they were stored at −80°C until processing. Sponge DNA was extracted as described previously (2, 5).Total sponge-derived genomic DNA was screened by PCR for the presence of poribacteria using a 16S rRNA gene primer set. Poribacterial 16S rRNA genes were amplified by employing a pair of Poribacteria-specific primers, POR389f (5′-ACG ATG CGA CGC CGC GTG-3′) and POR1130r (5′-GGC TCG TCA CCA GCG GTC-3′) (2). The poribacterial PCR products that were ca. 740 bp long derived from one sponge individual were cloned into the pGEM-T Easy vector (Promega, Madison, WI). Clone inserts were digested with restriction endonucleases MspI and HaeIII (New England Biolabs, Inc., United States), characterized to obtain restriction profiles and unique profiles, and sequenced. The compiled partial 16S rRNA gene sequences were then analyzed using BLASTN to select the most closely related poribacterial reference sequences.The sequences exhibiting levels of similarity of less than 97% were used for further analysis. Poribacterial 16S rRNA gene sequences were aligned using the ARB software package (7). The resulting alignment was imported into PAUP (10) and analyzed by using distance, maximum parsimony, and maximum likelihood algorithms together with bootstrap resamplings (3,000, 3,000, and 200 resamplings, respectively), and the resulting bootstrap values were applied to nodes on the ARB neighbor-joining tree. Signature sequences were detected using the ARB software package. A signature sequence is defined here as a short sequence that is present in a group of poribacterial sequences in a phylogenetic clade but is not found in any other clade in the poribacterial tree.Analysis of the 16S rRNA gene clone library sequences generated from sponge tissues revealed the presence of poribacteria in sponge individuals belonging to the orders Verongida, Astrophorida, Dictyoceratida, Haplosclerida, Lithistida, and Homosclerophorida, while poribacteria could not be detected in sponges belonging to the orders Hadromerida and Agelasida. In the order Halichondrida, poribacteria were detected in Xestospongia muta but not in Haliclona sp. Altogether, nine sponge species were added to the list of Poribacteria-containing sponges (Table ​(Table1).1). Three distinct clades were observed that were clearly supported by bootstrap values greater than 75 with every tree-building algorithm applied (Fig. ​(Fig.1),1), and one clade (clade I) was supported by bootstrap values of 64, 98, and 71 in distance, maximum parsimony, and maximum likelihood trees, respectively. Similarity calculations using approximately 740-bp amplified poribacterial 16S rRNA gene fragments and other poribacterial sequences from the NCBI database showed that the dissimilarity between clades was consistent with their separation in phylogenetic trees. For example, the levels of dissimilarity between members of clade I and clade II were 3 to 8%, while the levels of dissimilarity between members of clades I and III and between members of clades I and IV were 10 to 14% and 11 to 15%, respectively.Open in a separate windowFIG. 1.Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene, showing relationships of poribacterial clones from different global regions. The poribacterial clones on the right are additional clones belonging to the same clades as strains in the tree at the same level. Bootstrap confidence values of >75% for distance, maximum parsimony, and maximum likelihood algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina aerophoba; C, Aplysina cavernicola; F, Aplysina fistularis; L, Aplysina lacunosa; S, Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG, Rhabdastrella globostellata; T, Theonella swinhoei; X, Xestospongia muta. Scale bar = 0.1 nucleotide substitution per site.

TABLE 1.

Distribution of poribacteria in different demosponge orders

Use of a Mixture of Surrogates for Infectious Bioagents in a Standard Approach to Assessing Disinfection of Environmental Surfaces

We used a mixture of surrogates (Acinetobacter baumannii, Mycobacterium terrae, hepatitis A virus, and spores of Geobacillus stearothermophilus) for bioagents in a standardized approach to test environmental surface disinfectants. Each carrier containing 10 μl of mixture received 50 μl of a test chemical or saline at 22 ± 2°C. Disinfectant efficacy criteria were ≥6 log₁₀ reduction for the bacteria and the spores and ≥3 log₁₀ reduction for the virus. Peracetic acid (1,000 ppm) was effective in 5 min against the two bacteria and the spores but not against the virus. Chlorine dioxide (CD; 500 and 1,000 ppm) and domestic bleach (DB; 2,500, 3,500, and 5,000 ppm) were effective in 5 min, except for sporicidal activity, which needed 20 min of contact with either 1,000 ppm of CD or the two higher concentrations of DB.Disinfectant testing with a single type of organism does not represent field conditions, where bioagents or other pathogens may be mixed with other contaminants. Such an approach also cannot predict the true spectrum of microbicidal activity of a given chemical, while the identity of the target pathogen(s) is often unknown. We used a mixture of Acinetobacter baumannii, Mycobacterium terrae (15), hepatitis A virus (HAV) (4), and the spores of Geobacillus stearothermophilus as surrogates for infectious bioagents, with an added soil load on disks (1 cm in diameter; 0.75 mm thick) of brushed stainless steel (AISI no. 430; Muzeen & Blythe, Winnipeg, MB, Canada), to better simulate environmental surface disinfection (1, 11). Table ​Table11 gives details on the microbial strains, media used for their culture and recovery, and methods for preparing working stocks. The quantitative carrier test (QCT) method, ASTM standard E-2197 (1), was used to test the organisms singly and in a mixture. Each 200 μl of the inoculum contained 34 μl each of the four organisms, 40 μl of bovine mucin, 14 μl of yeast extract, and 10 μl of bovine serum albumin stocks.

TABLE 1.

Organisms in the mixture and their growth/recovery media and titers

Environmental Isolates of Burkholderia pseudomallei in Ceará State,Northeastern Brazil

Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.Melioidosis is an endemic disease in Southeast Asia and northern Australia (2, 4) and also occurs sporadically in other parts of the world (3, 7). Human melioidosis was reported to occur in Brazil only in 2003, when a family outbreak afflicted four sisters in the rural part of the municipality of Tejuçuoca, Ceará state (14). After this episode, there was one reported case of melioidosis in 2004 in the rural area of Banabuiú, Ceará (14). And in 2005, a case of melioidosis associated with near drowning after a car accident was confirmed to occur in Aracoiaba, Ceará (11).The goal of this study was to investigate the Tejuçuoca and Banabuiú municipalities, where human cases of melioidosis have been confirmed to occur, and to gain a better understanding of the ecology of Burkholderia pseudomallei in this region.We chose as central points of the study the residences and surrounding areas of the melioidosis patients in the rural areas of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50′W) (Fig. ​(Fig.1).1). There are two well-defined seasons in each of these locations: one rainy (running from January to May) and one dry (from June to December). A total of 600 samples were collected at five sites in Tejuçuoca (T1, T2, T3, T4, and T5) and five in Banabuiú (B1, B2, B3, B4, and B5), distributed as follows (Fig. ​(Fig.2):2): backyards (B1 and T1), places shaded by trees (B2 and T2), water courses (B3 and T3), wet places (B4 and T4), and stock breeding areas (B5 and T5).Open in a separate windowFIG. 1.Municipalities of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50″W).Open in a separate windowFIG. 2.Soil sampling sites in Banabuiú and Tejuçuoca.Once a month for 12 months (a complete dry/rainy cycle), five samples were gathered at five different depths: at the surface and at 10, 20, 30 and 40 cm (Table ​(Table1).1). The samples were gathered according to the method used by Inglis et al. (9). Additionally, the sample processing and B. pseudomallei identification were carried out as previously reported (1, 8, 9).

TABLE 1.

Distribution of samples with isolates by site and soil depth

Bar-Coded Pyrosequencing of 16S rRNA Gene Amplicons Reveals Changes in Ileal Porcine Bacterial Communities Due to High Dietary Zinc Intake

Feeding high levels of zinc oxide to piglets significantly increased the relative abundance of ileal Weissella spp., Leuconostoc spp., and Streptococcus spp., reduced the occurrence of Sarcina spp. and Neisseria spp., and led to numerical increases of all Gram-negative facultative anaerobic genera. High dietary zinc oxide intake has a major impact on the porcine ileal bacterial composition.Zinc oxide (ZnO) is used as a feed additive for diarrhea prophylaxis in piglets (23). However, the mode of action of ZnO is not fully understood. Besides its effects on the host (10, 30, 31), high dietary zinc levels may affect the diversity of intestinal microbial communities (2, 11, 20). The prevention of postweaning diarrhea in piglets due to high dietary ZnO intake may not be directly related to a reduction of pathogenic E. coli (8) but, rather, to the diversity of the coliform community (15). Studies on the impact of high ZnO levels on the porcine ileal bacterial community are scarce but nevertheless important, as bacterial diarrhea is initiated in the small intestine (9, 17). The small intestine is a very complex habitat with many different factors shaping the bacterial community. Studies on the ecophysiology (22) and maturation of the porcine ileal microbiota (13, 27) indicate a drastic impact directly after weaning and a gradual decline of modifications during the following 2 weeks. Thus, the time point for analysis chosen in this study (14 days postweaning) does reflect a more stable period of the ileal porcine microbiota. In this study, we used bar-coded pyrosequencing of 16S rRNA genes to gain further insight into the mode of action of pharmacological levels of ZnO in the gastrointestinal tract of young pigs.Total DNA was extracted from the ileal digesta of 40- to 42-day-old piglets using a commercial kit (Qiagen stool kit; Qiagen, Hilden, Germany) and PCR amplified with unique bar-coded primer sets targeting the V1-to-V3 and the V6-to-V8 hypervariable regions (see the supplemental material for detailed methods). The rationale behind this approach was derived from the fact that no single “universal” primer pair can completely cover a complex bacterial habitat (4, 24, 32, 33). Furthermore, these studies also show that in silico information on the coverage of selected primer sets diverges from empirical results, and hence, two hypervariable regions were chosen in this study to maximize the detection of phylogenetically diverse bacterial groups.Equimolar dilutions of all samples were combined into one master sample. Pyrosequencing was performed by Agowa (Berlin, Germany) on a Roche genome sequencer FLX system using a Titanium series PicoTiterPlate. The resulting data files were uploaded to the MG-RAST server (http://metagenomics.nmpdr.org/) (19) and processed with its SEED software tool using the RDP database (5) as the reference database. After automated sequence analysis, all sequences with less than five identical reads per sample were deleted in order to increase the confidence of sequence reads and reduce bias from possible sequencing errors (12, 16). Thus, 0.43% of all sequences were not considered (1,882 of 433,302 sequences). These sequences were assigned to a total of 238 genera, of which most only occurred in a few samples (see the supplemental material). Furthermore, all unclassified sequences were removed (8.7%; 41,467 of 474,769 sequences). Due to the use of the RDP reference database, the SEED software incorrectly assigned the majority of unclassified sequences as unclassified Deferribacterales (83%; 34,393 sequences), which were actually identified as 16S soybean or wheat chloroplasts by BLAST or as cyanobacterial chloroplasts by the RDP II seqmatch tool.The pyrosequencing results for the two primer combinations were merged by taking only sequences from the primer combination that yielded the higher number of reads for a specific sequence assignment in a sample. The remaining reads were used to calculate the relative contribution of assigned sequences to total sequence reads in a sample.The Firmicutes phylum dominated the small intestinal bacterial communities in both the control group and the group with high dietary ZnO intake, with 98.3% and 97.0% of total sequence reads, respectively. No significant influence of high dietary ZnO intake was found for the main phyla Proteobacteria (0.92% versus 1.84%), Actinobacteria (0.61% versus 0.75%), Bacteroidetes (0.15% versus 0.17%), and Fusobacteria (0.09% versus 0.12%).On the order level, a total of 20 bacterial orders were detected (data not shown). Lactobacillales dominated bacterial communities in the control and high-dietary-ZnO-intake groups, with 83.37% and 93.24% of total reads. Lactic acid bacteria are well known to dominate the bacterial community in the ileum of piglets (11, 22). No significant difference between the control group and the group with high dietary ZnO intake was observed on the order level, although high dietary ZnO intake led to a strong numerical decrease for Clostridiales (14.4 ± 24.0% [mean ± standard deviation] versus 2.8 ± 1.7%), as well as to numerical increases for Pseudomonadales (0.3 ± 0.3% versus 0.6 ± 0.6%) and Enterobacteriales (0.2 ± 0.2% versus 0.5 ± 0.6%).On the genus level, a total of 103 genera were detected. Table ​Table11 summarizes the main 31 genera which exceeded 0.05% of total reads (see the supplemental material for a complete list). Lactobacilli clearly dominated the bacterial communities in both trial groups, but they also were numerically lower due to high dietary ZnO intake.

TABLE 1.

Bacterial genera in the ileum of piglets fed diets supplemented with 200 or 3,000 ppm ZnO

Detection and Quantification of the Coral Pathogen Vibrio coralliilyticus by Real-Time PCR with TaqMan Fluorescent Probes

A real-time quantitative PCR-based detection assay targeting the dnaJ gene (encoding heat shock protein 40) of the coral pathogen Vibrio coralliilyticus was developed. The assay is sensitive, detecting as little as 1 CFU per ml in seawater and 10⁴ CFU per cm² of coral tissue. Moreover, inhibition by DNA and cells derived from bacteria other than V. coralliilyticus was minimal. This assay represents a novel approach to coral disease diagnosis that will advance the field of coral disease research.Vibrio coralliilyticus has recently emerged as a coral pathogen of concern on reefs throughout the Indo-Pacific. It was first implicated as the etiological agent responsible for bleaching and tissue lysis of the coral Pocillopora damicornis on Zanzibar reefs (2). More recently, V. coralliilyticus has been identified as the causative agent of white syndrome (WS) outbreaks on several Pacific reefs (14). WS is a collective term describing coral diseases characterized by a spreading band of tissue loss exposing white skeleton on Indo-Pacific scleractinian corals (16). V. coralliilyticus is an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease (13) and therefore provides an ideal model for the development of diagnostic assays to detect coral disease. Current coral disease diagnostic methods, which are based primarily upon field-based observations of macroscopic disease signs, often detect disease only at the latest stages of infection, when control measures are least effective. The development of diagnostic tools targeting pathogens underlying coral disease pathologies may provide early indications of infection, aid the identification of disease vectors and reservoirs, and assist managers in developing strategies to prevent the spread of coral disease outbreaks. In this paper, we describe the development and validation of a TaqMan-based real-time quantitative PCR (qPCR) assay that targets a segment of the V. coralliilyticus heat shock protein 40-encoding gene (dnaJ).Nucleotide sequences of the dnaJ gene were retrieved from relevant Vibrio species, including V. coralliilyticus (LMG 20984), using the National Center for Biotechnology Information''s (NCBI) Entrez Nucleotide Database search tool (http://www.ncbi.nlm.nih.gov/). Gene sequences of strains not available in public databases (V. coralliilyticus strains LMG 21348, LMG 21349, LMG 21350, LMG 10953, LMG 20538, LMG 23696, LMG 23691, LMG 23693, LMG 23692, and LMG 23694) were obtained through extraction of total DNA using a Promega Wizard Prep DNA Purification Kit (Promega, Sydney, Australia), PCR amplification, and sequencing using primers and thermal cycling parameters described by Nhung et al. (8). A 128-bp region (nucleotides 363 to 490) containing high concentrations of single nucleotide polymorphisms (SNPs), which were conserved within V. coralliilyticus strains but differed from non-V. coralliilyticus strains, was identified, and oligonucleotide primers Vc_dnaJ_F1 (5′-CGG TTC GYG GTG TTT CAA AA-3′) and Vc_dnaJ_R1 (5′-AAC CTG ACC ATG ACC GTG ACA-3′) and a TaqMan probe, Vc_dnaJ_TMP (5′-6-FAM-CAG TGG CGC GAA G-MGBNFQ-3′; 6-FAM is 6-carboxyfluorescein and MGBNFQ is molecular groove binding nonfluorescent quencher), were designed to target this region. The qPCR assay was optimized and validated using DNA extracted from V. coralliilyticus isolates, nontarget Vibrio species, and other bacterial species grown in marine broth (MB) (Table ​(Table1),1), under the following optimal conditions: 1× TaqMan buffer A, 0.5 U of AmpliTaq Gold DNA polymerase, 200 μM deoxynucleotide triphosphates (with 400 μM dUTP replacing deoxythymidine triphosphate), 0.2 U of AmpErase uracil N-glycosylase (UNG), 3 mM MgCl₂, 0.6 μM each primer, 0.2 μM fluorophore-labeled TaqMan, 1 μl of template, and sterile MilliQ water for a total reaction volume to 20 μl. All assays were conducted on a RotoGene 300 (Corbett Research, Sydney, Australia) real-time analyzer with the following cycling parameters: 50°C for 120 s (UNG activation) and 95°C for 10 min (AmpliTaq Gold DNA polymerase activation), followed by 40 cycles of 95°C for 15 s (denaturation) and 60°C for 60 s (annealing/extension). During the annealing/extension phase of each thermal cycle, fluorescence was measured in the FAM channel (470-nm excitation and 510-nm detection).

TABLE 1.

Species, strain, and threshold cycle for all bacterial strains tested^a

Quantitative Fluorescence In Situ Hybridization of Microbial Communities in the Rumens of Cattle Fed Different Diets

At present there is little quantitative information on the identity and composition of bacterial populations in the rumen microbial community. Quantitative fluorescence in situ hybridization using newly designed oligonucleotide probes was applied to identify the microbial populations in liquid and solid fractions of rumen digesta from cows fed barley silage or grass hay diets with or without flaxseed. Bacteroidetes, Firmicutes, and Proteobacteria were abundant in both fractions, constituting 31.8 to 87.3% of the total cell numbers. They belong mainly to the order Bacteroidales (0.1 to 19.2%), hybridizing with probe BAC1080; the families Lachnospiraceae (9.3 to 25.5%) and Ruminococcaceae (5.5 to 23.8%), hybridizing with LAC435 and RUM831, respectively; and the classes Deltaproteobacteria (5.8 to 28.3%) and Gammaproteobacteria (1.2 to 8.2%). All were more abundant in the rumen communities of cows fed diets containing silage (75.2 to 87.3%) than in those of cows fed diets containing hay (31.8 to 49.5%). The addition of flaxseed reduced their abundance in the rumens of cows fed silage-based diets (to 45.2 to 58.7%) but did not change markedly their abundance in the rumens of cows fed hay-based diets (31.8 to 49.5%). Fibrolytic species, including Fibrobacter succinogenes and Ruminococcus spp., and archaeal methanogens accounted for only a small proportion (0.4 to 2.1% and 0.2 to 0.6%, respectively) of total cell numbers. Depending on diet, between 37.0 and 91.6% of microbial cells specifically hybridized with the probes used in this study, allowing them to be identified in situ. The identities of other microbial populations (8.4 to 63.0%) remain unknown.The rumen is an anaerobic ecosystem used by herbivores to convert fibrous plant material into fermentation products that are in turn used as energy by the host. Fibrolytic degradation is accomplished by a complex microbial community which includes specialized fungi, protozoa, and bacteria (14). More than 200 bacterial species (5) have been isolated from rumen, and many of these have been phylogenetically and physiologically characterized. Several of these, including Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, have the ability to hydrolyze cellulose in axenic culture (24). Despite the presence of these fibrolytic populations, a large portion of the fiber in low-quality forage diets passes through the rumen undigested. In the rumen, fibrolytic bacteria do not digest plant cell walls in isolation but rather interact with a consortium of bacteria (18). Although culture-dependent studies have improved our understanding of rumen microbiology, the importance of the isolates to the structure and function of the rumen microbial community, with the possible exception of the fibrolytic strains, is still unknown. Expanding our knowledge of the structure and function of the rumen microbial community may provide insights into approaches to improve the efficiency of fiber digestion and biofuel production (14).To provide a high-resolution view of the population structure of the rumen bacterial community, we used quantitative fluorescence in situ hybridization (qFISH) to investigate the composition and distribution of bacterial populations associated with the liquid and solid rumen contents from 12 ruminally cannulated Holstein dairy cows (3 cows were used for each diet) fed (for at least 21 days) grass hay or barley silage diets with or without flaxseed (Table ​(Table1).1). Six new 16S rRNA-targeted FISH probes (Table ​(Table2)2) for not only the fibrolytic groups but also other unclassified bacterial groups in the rumen were designed, using ARB software (17), against the rumen 16S rRNA gene sequences (data not shown) retrieved from the Ribosomal Database Project (RDP) database (6). The new probes target Bacteroidales-related clones (probe BAC1080) (phylum Bacteroidetes), Lachnospiraceae- and Ruminococcaceae-related clones (probes LAC435 and RUM831, respectively) (phylum Firmicutes), Butyrivibrio fibrisolvens-related clones (probe BFI826), and R. albus- and R. flavefaciens-related clones (probes RAL1436 and RFL155, respectively).

TABLE 1.

Composition of diets used in this study

Physicochemical Basis for the Inhibitory Effects of Organic and Inorganic Salts on the Growth of Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum

Twenty-one salts were tested for their effects on the growth of Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum. In liquid medium, 11 salts (0.2 M) exhibited strong inhibition of bacterial growth. The inhibitory action of salts relates to the water-ionizing capacity and the lipophilicity of their constituent ions.Different biochemical mechanisms have been put forth to explain the antimicrobial activity of organic and inorganic salts, including inhibition of several steps of the energy metabolism (benzoate, bicarbonate, propionate, sorbate, and sulfite salts) (2, 3, 11, 16, 17, 19, 25) and complexation to DNA and RNA (aluminum and sulfites) (12, 13, 15, 20, 27, 28). However, little is known about the physicochemical basis for the general antimicrobial action of salts. The objective of this work was to gain an understanding of the relationship between the inhibitory action of salts on bacterial growth and their physicochemical properties by using the bacteria Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora) and Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica). These bacteria are responsible for soft rot, a disease of economic importance affecting numerous stored vegetable crops (14, 22).Pectobacterium carotovorum subsp. carotovorum (strain Ecc 1367) and P. atrosepticum (strain Eca 709), provided by the Laboratoire de Diagnostic en Phytoprotection (MAPAQ, Québec, Canada), were grown in 250-ml flasks containing 50 ml of 20% tryptic soy broth (Difco Laboratories, Becton Dickinson, Sparks, MD) amended with salts (200 mM) or unamended (control), by incubation at 24°C with agitation (150 rpm; Lab-Line Instruments Inc., Melrose Park, IL) for 24 h. The pHs of the media were not adjusted but varied with the type of salts, unless stated otherwise. Flasks were inoculated with 100 μl of each bacterial suspension (1 × 10⁷ CFU/ml). Bacterial growth was determined by turbidimetry at 600 nm with a UV/visible spectrophotometer (Ultrospec 2000; Pharmacia Biotech Ltd, Cambridge, United Kingdom), using appropriate blanks. Results were expressed as the percentage of growth inhibition compared with the growth of the control. A completely randomized experimental design with three replicates was used, the experimental unit being a flask. Analysis of variance was carried out with the GLM (general linear model) procedure of SAS (SAS Institute, Cary, NC) software. When they were significant (P < 0.05), treatment means were compared using Fisher''s protected least-significant-difference test.Among the 21 salts tested, sodium carbonate, sodium metabisulfite, trisodium phosphate, aluminum lactate, aluminum chloride, sodium bicarbonate, sodium propionate, ammonium acetate, aluminum dihydroxy acetate, potassium sorbate, and sodium benzoate exhibited strong inhibition (≥97%) of the growth of both P. carotovorum subsp. carotovorum and P. atrosepticum (Table ​(Table1).1). Calcium chloride, sodium formate, sodium acetate, ammonium hydrogen phosphate, and sodium hydrogen phosphate exhibited a moderately inhibitory effect; sodium lactate and tartrate had no effect. On the other hand, ammonium chloride, potassium chloride, and sodium chloride stimulated the growth of P. atrosepticum.

TABLE 1.

Effect of salts on the growth of P. atrosepticum and P. carotovorum subsp. carotovorum

Characterization of a Thermostable Short-Chain Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus sibiricus

Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.Alcohol dehydrogenases (ADHs; EC 1.1.1.1.) catalyze the interconversion of alcohols to their corresponding aldehydes or ketones by using different redox-mediating cofactors. NAD(P)-dependent ADHs, due to their broad substrate specificity and enantioselectivity, have attracted particular attention as catalysts in industrial processes (5). However, mesophilic ADHs are unstable at high temperatures, sensitive to organic solvents, and often lose activity during immobilization. In this relation, there is a considerable interest in ADHs from extremophilic microorganisms; among them, Archaea are of great interest. The representatives of all groups of NAD(P)-dependent ADHs have been detected in genomes of Archaea (11, 12); however, only a few enzymes have been characterized, and the great majority of them belong to medium-chain (3, 4, 14, 16, 19) or long-chain iron-activated ADHs (1, 8, 9). Up to now, a single short-chain archaeal ADH from Pyrococcus furiosus (10, 18) and only one archaeal aldo-keto reductase also from P. furiosus (11) have been characterized.Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a high-temperature oil reservoir capable of growth on complex organic substrates (15). The complete genome sequence of T. sibiricus has been recently determined and annotated (13). Several ADHs are encoded by the T. sibiricus genome, including three short-chain ADHs (Tsib_0319, Tsib_0703, and Tsib_1998) (13). In this report, we describe the cloning and expression of the Tsib_0319 gene from T. sibiricus and the purification and the biochemical characterization of its product, the thermostable short-chain ADH (TsAdh319).The Tsib_0319 gene encodes a protein with a size of 234 amino acids and the calculated molecular mass of 26.2 kDa. TsAdh319 has an 85% degree of sequence identity with short-chain ADH from P. furiosus (AdhA; PF_0074) (18). Besides AdhA, close homologs of TsAdh319 were found among different bacterial ADHs, but not archaeal ADHs. The gene flanked by the XhoI and BamHI sites was PCR amplified using two primers (sense primer, 5′-GTTCTCGAGATGAAGGTTGCTGTGATAACAGGG-3′, and antisense primer, 5′-GCTGGATCCTCAGTATTCTGGTCTCTGGTAGACGG-3′) and cloned into the pET-15b vector. TsAdh319 was overexpressed, with an N-terminal His₆ tag in Escherichia coli Rosetta-gami (DE3) and purified to homogeneity by metallochelating chromatography (Hi-Trap chelating HP column; GE Healthcare) followed by gel filtration on Superdex 200 10/300 GL column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl. The homogeneity and the correspondence to the calculated molecular mass of 28.7 kDa were verified by SDS-PAGE (7). The molecular mass of native TsAdh319 was 56 to 60 kDa, which confirmed the dimeric structure in solution.The standard ADH activity measurement was made spectrophotometrically at the optimal pH by following either the reduction of NADP (in 50 mM Gly-NaOH buffer; pH 10.5) or the oxidation of NADPH (in 0.1 M sodium phosphate buffer; pH 7.5) at 340 nm at 60°C. The enzyme exhibited a strong preference for NADP(H) and broad substrate specificity (Table ​(Table1).1). The highest oxidation rates were found with pentoses d-arabinose (2.0 U mg⁻¹) and d-xylose (2.46 U mg⁻¹), and the highest reduction rates were found with dimethylglyoxal (5.9 U mg⁻¹) and pyruvaldehyde (2.2 U mg⁻¹). The enzyme did not reduce sugars which were good substrates for the oxidation reaction. The kinetic parameters of TsAdh319 determined for the preferred substrates are shown in Table ​Table2.2. The enantioselectivity of the enzyme was estimated by measuring the conversion rates of 2-butanol enantiomers. TsAdh319 showed an evident preference, >2-fold, for (S)-2-butanol over (RS)-2-butanol. The enzyme stereoselectivity is confirmed by the preferred oxidation of d-arabinose over l-arabinose (Table ​(Table1).1). The fact that TsAdh319 is metal independent was supported by the absence of a significant effect of TsAdh319 preincubation with 10 mM Me²⁺ for 30 min before measuring the activity in the presence of 1 mM Me²⁺ or EDTA (Table ​(Table3).3). TsAdh319 also exhibited a halophilic property, so the enzyme activity increased in the presence of NaCl and KCl and the activation was maintained even at concentration of 4 M and 3 M, respectively (Table ​(Table33).

TABLE 1.

Substrate specificity of TsAdh319