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

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

Economic Benefits of Investing in Women’s Health: A Systematic Review

Background

Globally, the status of women’s health falls short of its potential. In addition to the deleterious ethical and human rights implications of this deficit, the negative economic impact may also be consequential, but these mechanisms are poorly understood. Building on the literature that highlights health as a driver of economic growth and poverty alleviation, we aim to systematically investigate the broader economic benefits of investing in women’s health.

Methods

Using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, we systematically reviewed health, gender, and economic literature to identify studies that investigate the impact of women’s health on micro- and macroeconomic outcomes. We developed an extensive search algorithm and conducted searches using 10 unique databases spanning the timeframe 01/01/1970 to 01/04/2013. Articles were included if they reported on economic impacts stemming from changes in women’s health (table of outcome measures included in full review,

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

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

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)

Widespread Distribution of Cell Defense against d-Aminoacyl-tRNAs

Several l-aminoacyl-tRNA synthetases can transfer a d-amino acid onto their cognate tRNA(s). This harmful reaction is counteracted by the enzyme d-aminoacyl-tRNA deacylase. Two distinct deacylases were already identified in bacteria (DTD1) and in archaea (DTD2), respectively. Evidence was given that DTD1 homologs also exist in nearly all eukaryotes, whereas DTD2 homologs occur in plants. On the other hand, several bacteria, including most cyanobacteria, lack genes encoding a DTD1 homolog. Here we show that Synechocystis sp. PCC6803 produces a third type of deacylase (DTD3). Inactivation of the corresponding gene (dtd3) renders the growth of Synechocystis sp. hypersensitive to the presence of d-tyrosine. Based on the available genomes, DTD3-like proteins are predicted to occur in all cyanobacteria. Moreover, one or several dtd3-like genes can be recognized in all cellular types, arguing in favor of the nearubiquity of an enzymatic function involved in the defense of translational systems against invasion by d-amino acids.Although they are detected in various living organisms (reviewed in Ref. 1), d-amino acids are thought not to be incorporated into proteins, because of the stereospecificity of aminoacyl-tRNA synthetases and of the translational machinery, including EF-Tu and the ribosome (2). However, the discrimination between l- and d-amino acids by aminoacyl-tRNA synthetases is not equal to 100%. Significant d-aminoacylation of their cognate tRNAs by Escherichia coli tyrosyl-, tryptophanyl-, aspartyl-, lysyl-, and histidyl-tRNA synthetases has been characterized in vitro (3–9). Recently, using a bacterium, transfer of d-tyrosine onto tRNA^Tyr was shown to occur in vivo (10).With such misacylation reactions, the resulting d-aminoacyl-tRNAs form a pool of metabolically inactive molecules, at best. At worst, d-aminoacylated tRNAs infiltrate the protein synthesis machinery. Although the latter harmful possibility has not yet been firmly established, several cells were shown to possess a d-tyrosyl-tRNA deacylase, or DTD, that should help them counteract the accumulation of d-aminoacyl-tRNAs. This enzyme shows a broad specificity, being able to remove various d-aminoacyl moieties from the 3′-end of a tRNA (4–6, 11). Such a function makes the deacylase a member of the family of enzymes capable of editing in trans mis-aminoacylated tRNAs. This family includes several homologs of aminoacyl-tRNA synthetase editing domains (12), as well as peptidyl-tRNA hydrolase (13, 14).Two distinct deacylases have already been discovered. The first one, called DTD1, is predicted to occur in most bacteria and eukaryotes (see d-amino acids, including d-tyrosine (6). In fact, in an E. coli Δdtd strain grown in the presence of 2.4 mm d-tyrosine, as much as 40% of the cellular tRNA^Tyr pool becomes esterified with d-tyrosine (10).

TABLE 1

Distribution of DTD1 and DTD2 homologs in various phylogenetic groupsHomologs of DTD1 and DTD2 were searched for using a genomic Blast analysis against complete genomes in the NCBI Database (www.ncbi.nlm.nih.gov). Values in the table are number of species. For instance, E. coli is counted only once in γ-proteobacteria despite the fact that several E. coli strains have been sequenced.

Isolation of VanA-Type Vancomycin-Resistant Enterococcus Strains from Domestic Poultry Products with Enrichment by Incubation in Buffered Peptone Water at 42°C

Eight VanA-type enterococcal strains were isolated from 8 of 171 domestic poultry products by using enrichment by incubation in buffered peptone water at 35°C and 42°C. The pulsed-field gel electrophoresis patterns of all six VanA-type Enterococcus faecalis isolates were nearly indistinguishable, indicating the presence of a specific clone in Japan.The use of avoparcin as a growth promoter and prophylactic agent in feed has been associated with the occurrence of vancomycin-resistant enterococci (VRE) in farm animals in the European Union (EU) (2, 3, 4, 11). In Japan, avoparcin was also used as a food additive on animal farms for approximately 7 years until it was banned in March 1997 (10, 22). However, there has been no documentation in the literature on the isolation of VanA-type VRE from domestic poultry products in Japan (8, 10). Several traditional methods for detecting enterococci, including VRE, in poultry products include an enrichment step using buffered peptone water (BPW) (7, 12, 14, 21). Although BPW is typically incubated at 35 to 37°C, based on the reported optimal growth temperature for enterococci of 35°C (19, 20), the growth of VRE at this temperature can be inhibited by antagonistic activities of background microflora in samples contaminated with low levels of VRE, as 35 to 37°C is also the optimal growth temperature for most mesophilic microorganisms. Several investigators have demonstrated that incubation at an elevated temperature using selective enrichment medium was useful in the isolation of enterococcal strains from retail meat (9, 17, 18). To our knowledge, however, no study has evaluated cultivation temperature during the enrichment process in the isolation of VanA-type VRE from poultry products to examine the influence of background microorganisms on VanA-type VRE growth in BPW. In this study, we carried out isolation of VanA-type VRE from retail Japanese domestic chicken products by using two different enrichment temperatures (35°C and 42°C) and characterized VanA-type VRE isolates by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). We also assessed the effects of incubation temperature on the growth of VanA-type VRE under pure culture conditions, on the rates of recovery of inoculated VanA-type VRE strains from artificially contaminated poultry samples, and on the inhibition of suspected enterococcal strains by background microorganisms in poultry samples.A total of 171 retail domestic poultry products obtained in Osaka, Japan, between September 2008 and April 2009 were examined for the presence of VanA-type VRE. Twenty-five grams of each sample was removed aseptically and placed in a sterile plastic bag (30 by 19 cm), to which 225 ml of BPW (Eiken Chemical Co., Ltd., Tokyo, Japan) was added. The sample was then incubated for 24 h at either 35°C or 42°C. Ten microliters of the culture supernatants was spread plated on Enterococcosel agar (Becton, Dickinson and Company, Sparks, MD) plates containing 4 μg/ml vancomycin (Wako Pure Chemical Industries, Ltd., Hyogo, Japan) and incubated at 36°C for 48 h. Screening for the vanA gene was done using the colony-sweep PCR method with phosphate-buffered saline (PBS) washed-cell suspension, Z Taq polymerase (Takara Biochemicals, Shiga, Japan), and previously described primers (1, 16). PCR-positive sweeps were streaked for isolated VanA-type VRE colonies on an Enterococcosel agar plate containing 32 μg/ml vancomycin. The plates were incubated at 36°C for 48 h, and suspected VRE colonies were confirmed as enterococci by conventional biochemical methods (8). In addition, identification of Enterococcus faecalis and Enterococcus faecium harboring the vanA gene was performed by a multiple PCR assay previously described by Depardieu et al. (6). For the five VanA-positive poultry product samples obtained in October 2008 and April 2009, the number of VanA-type VRE was determined by plating 1-ml volumes of samples diluted 10-fold in sterile peptone saline onto Enterococcosel agar plates containing 32 μg/ml vancomycin. Colonies were counted after incubation at 36°C for 48 h. MICs of vanA-genotype VRE isolates to vancomycin and teicoplanin were determined using the Etest method (AB Biodisk, Solna, Sweden) in accordance with the criteria of the Clinical and Laboratory Standards Institute (5). Subtyping of VanA-type VRE isolates was performed by PFGE with SmaI-digested chromosomal DNA (8, 15). PFGE was performed on a 1.0% SeaKem Gold agarose gel (Cambrex Bio Science, Rockland, ME) and a CHEF-DRIII apparatus (Bio-Rad Laboratories, Hercules, CA). The PFGE standard strain Salmonella enterica serovar Braenderup H9812 was obtained from the PulseNet program, Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, through the National Institute of Infectious Diseases (Japan).The two representative VanA-type VRE strains were precultured overnight in Trypticase soy broth (Becton, Dickinson and Company) at 36°C. Each culture medium was diluted in BPW to obtain 2 log CFU/ml, and 100 μl of the culture dilution was inoculated into 40 ml of brain heart infusion (BHI) broth (Becton, Dickinson and Company) in a 200-ml Erlenmeyer flask. The flask was incubated at 35°C or 42°C with constant shaking (160 rpm) for 24 h, and the optical density at 600 nm (OD₆₀₀) of the cultures was monitored every 30 min using an OD-Monitor A & S (Taitec Co., Ltd., Saitama, Japan). Each strain was examined in triplicate. All VanA-type isolates in this study were inoculated onto the poultry samples to compare the effect of BPW incubation at 42°C on the isolation of VanA-type VRE with the effect of incubation at 35°C. The strains were precultured at 36°C for 24 h under static conditions and then serially diluted in BPW to obtain a cell concentration of approximately 3 log CFU/ml. One hundred microliters of each bacterial cell suspension was individually inoculated onto two 25-g portions of each poultry sample in sterile plastic bags. Each inoculated poultry sample was added to 225 ml of BPW and homogenized for 1 min. One sample was incubated at 35°C for 24 h, and the second was incubated at 42°C for 24 h. Colony-sweep PCR analyses with Enterococcosel agar containing 4 μg/ml vancomycin for the presence of the vanA gene were then performed as described above. The artificially contaminated poultry sample experiment was performed in triplicate. To evaluate the success of cultivation using the different enrichment temperatures, data regarding the identification of the vanA gene from the colony-sweep PCR were analyzed using Fisher''s exact test. To examine the effect of enrichment temperature on inhibition of suspected enterococcal strains by background microflora, growth kinetics were compared among these microorganisms in three different poultry samples. For each sample, two 25-g portions were placed into sterile plastic bags to which 225 ml of BPW was added. After homogenization, the samples were incubated at 35°C and 42°C. Each culture was sampled every 3 h for the initial 15 h and after incubation for 24 h. The number of background microorganisms was determined by plating 1-ml volumes of samples serially diluted in PBS onto standard agar (Eiken) plates. Colonies were counted after incubation at 36°C for 24 h. The growth of enterococcal strains was monitored by plating 100-μl volumes of appropriate dilutions onto EF agar plates (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and characteristic enterococcal colonies were counted after incubation at 36°C for 48 h.Colony-sweep PCR analyses revealed that 8 of 171 domestic poultry samples were positive for the vanA gene. Isolation of VRE strains from the PCR-positive sweeps resulted in the identification of six vanA-genotype E. faecalis strains and two vanA-genotype E. faecium strains. Of the eight vanA-genotype VRE-positive samples, five were identified from only the 42°C BPW enrichment culture, while only a single isolate was uniquely identified from incubation at 35°C (Table ​(Table1).1). The number of VanA-type VRE was beneath the detection limit (i.e., less than 10 CFU/g) in each of the five VRE-positive poultry samples obtained in October 2008 and April 2009, suggesting that VanA-type VRE levels might be markedly low in domestic chickens in Japan. All vanA-genotype E. faecalis and E. faecium strains had vancomycin and teicoplanin MICs of >256 μg/ml and ≥32 μg/ml, respectively, confirming that these strains corresponded to the VanA phenotype (Table ​(Table1).1). Although the PFGE patterns of two VanA-type E. faecium strains were distinct (Fig. ​(Fig.1,1, lanes 7 and 8), the patterns of all E. faecalis isolates were almost indistinguishable (lanes 1 to 6). Given that the six VanA-type E. faecalis strains were isolated from distinct poultry samples that were obtained from 5 different retail shops, these results indicated that a specific E. faecalis clone harboring the vanA gene may be widely disseminated in Japan. The dominant species of VanA-type VRE isolated from poultry sources has been shown to differ depending on region. In New Zealand, greater than 80% of VRE isolates from broiler fecal samples were identified as vanA-genotype E. faecalis strains with very similar PFGE patterns, indicating that once established, a single, dominant clonal strain is capable of widespread contamination, and supporting the idea of clonal VRE dissemination rather than horizontal transfer of vancomycin resistance elements (13). Our findings also suggest that the epidemiological features of VanA-type VRE strains associated with poultry production environments in Japan may be similar to those observed in New Zealand. To our knowledge, this is the first report of the isolation of VanA-type VRE from retail domestic poultry products in Japan.Open in a separate windowFIG. 1.DNA fingerprinting of VanA-type VRE (E. faecalis and E. faecium strains) isolated from retail domestic poultry samples. Isolated genomic DNA of each isolate was digested with SmaI and subjected to PFGE. Lane 1, E. faecalis Ha8; lane 2, E. faecalis Ha16; lane 3, E. faecalis Ha36; lane 4, E. faecalis Ha55; lane 5, E. faecalis Ha58; lane 6, E. faecalis Ha61; lane 7, E. faecium Ha20; lane 8, E. faecium Ha26; lanes M, XbaI-digested PFGE patterns of DNA size standard Salmonella enterica serovar Braenderup H9812. Numbers to the left of the gel image indicate the molecular size in kilobase pairs.

TABLE 1.

Characteristics of vancomycin-resistant enterococcal strains isolated from domestic poultry samples in this study

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

Variation in Adult Plant Phenotypes and Partitioning among Seed and Stem-Borne Roots across Brachypodium distachyon Accessions to Exploit in Breeding Cereals for Well-Watered and Drought Environments

Seedling roots enable plant establishment. Their small phenotypes are measured routinely. Adult root systems are relevant to yield and efficiency, but phenotyping is challenging. Root length exceeds the volume of most pots. Field studies measure partial adult root systems through coring or use seedling roots as adult surrogates. Here, we phenotyped 79 diverse lines of the small grass model Brachypodium distachyon to adults in 50-cm-long tubes of soil with irrigation; a subset of 16 lines was droughted. Variation was large (total biomass, ×8; total root length [TRL], ×10; and root mass ratio, ×6), repeatable, and attributable to genetic factors (heritabilities ranged from approximately 50% for root growth to 82% for partitioning phenotypes). Lines were dissected into seed-borne tissues (stem and primary seminal axile roots) and stem-borne tissues (tillers and coleoptile and leaf node axile roots) plus branch roots. All lines developed one seminal root that varied, with branch roots, from 31% to 90% of TRL in the well-watered condition. With drought, 100% of TRL was seminal, regardless of line because nodal roots were almost always inhibited in drying topsoil. Irrigation stimulated nodal roots depending on genotype. Shoot size and tillers correlated positively with roots with irrigation, but partitioning depended on genotype and was plastic with drought. Adult root systems of B. distachyon have genetic variation to exploit to increase cereal yields through genes associated with partitioning among roots and their responsiveness to irrigation. Whole-plant phenotypes could enhance gain for droughted environments because root and shoot traits are coselected.Adult plant root systems are relevant to the size and efficiency of seed yield. They supply water and nutrients for the plant to acquire biomass, which is positively correlated to the harvest index (allocation to seed grain), and the stages of flowering and grain development. Modeling in wheat (Triticum aestivum) suggested that an extra 10 mm of water absorbed by such adult root systems during grain filling resulted in an increase of approximately 500 kg grain ha⁻¹ (Manschadi et al., 2006). This was 25% above the average annual yield of wheat in rain-fed environments of Australia. This number was remarkably close to experimental data obtained in the field in Australia (Kirkegaard et al., 2007). Together, these modeling and field experiments have shown that adult root systems are critical for water absorption and grain yield in cereals, such as wheat, emphasizing the importance of characterizing adult root systems to identify phenotypes for productivity improvements.Most root phenotypes, however, have been described for seedling roots. Seedling roots are essential for plant establishment, and hence, the plant’s potential to set seed. For technical reasons, seedlings are more often screened than adult plants because of the ease of handling smaller plants and the high throughput. Seedling-stage phenotyping may also improve overall reproducibility of results because often, growth media are soil free. Seedling soil-free root phenotyping conditions are well suited to dissecting fine and sensitive mechanisms, such as lateral root initiation (Casimiro et al., 2003; Péret et al., 2009a, 2009b). A number of genes underlying root processes have been identified or characterized using seedlings, notably with the dicotyledonous models Arabidopsis (Arabidopsis thaliana; Mouchel et al., 2004; Fitz Gerald et al., 2006; Yokawa et al., 2013) and Medicago truncatula (Laffont et al., 2010) and the cereals maize (Zea mays; Hochholdinger et al., 2001) and rice (Oryza sativa; Inukai et al., 2005; Kitomi et al., 2008).Extrapolation from seedling to adult root systems presents major questions (Hochholdinger and Zimmermann, 2008; Chochois et al., 2012; Rich and Watt, 2013). Are phenotypes in seedling roots present in adult roots given developmental events associated with aging? Is expression of phenotypes correlated in seedling and adult roots if time compounds effects of growth rates and growth conditions on roots? Watt et al. (2013) showed in wheat seedlings that root traits in the laboratory and field correlated positively but that neither correlated with adult root traits in the field. Factors between seedling and adult roots seemed to be differences in developmental stage and the time that growing roots experience the environment.Seedling and adult root differences may be larger in grasses than dicotyledons. Grass root systems have two developmental components: seed-borne (seminal) roots, of which a number emerge at germination and continue to grow and branch throughout the plant life, and stem-borne (nodal or adventitious) roots, which emerge from around the three-leaf stage and continue to emerge, grow, and branch throughout the plant life. Phenotypes and traits of adult root systems of grasses, which include the major cereal crops wheat, rice, and maize, are difficult to predict in seedling screens and ideally identified from adult root systems first (Gamuyao et al., 2012).Phenotyping of adult roots is possible in the field using trenches (Maeght et al., 2013) or coring (Wasson et al., 2014). A portion of the root system is captured with these methods. Alternatively, entire adult root systems can be contained within pots dug into the ground before sowing. These need to be large; field wheat roots, for example, can reach depths greater than 1.5 m depending on genotype and environment. This method prevents root-root interactions that occur under normal field sowing of a plant canopy and is also a compromise.A solution to the problem of phenotyping adult cereal root systems is a model for monocotyledon grasses: Brachypodium distachyon. B. distachyon is a small-stature grass with a small genome that is fully sequenced (Vogel et al., 2010). It has molecular tools equivalent to those available in Arabidopsis (Draper et al., 2001; Brkljacic et al., 2011; Mur et al., 2011). The root system of B. distachyon reference line Bd21 is more similar to wheat than other model and crop grasses (Watt et al., 2009). It has a seed-borne primary seminal root (PSR) that emerges from the embryo at seed germination and multiple stem-borne coleoptile node axile roots (CNRs) and leaf node axile roots (LNRs), also known as crown roots or adventitious roots, that emerge at about three leaves through to grain development. Branch roots emerge from all root types. There are no known anatomical differences between root types of wheat and B. distachyon (Watt et al., 2009). In a recent study, we report postflowering root growth in B. distachyon line Bd21-3, showing that this model can be used to answer questions relevant to the adult root systems of grasses (Chochois et al., 2012).In this study, we used B. distachyon to identify adult plant phenotypes related to the partitioning among seed-borne and stem-borne shoots and roots for the genetic improvement of well-watered and droughted cereals (Fig. 1; Krassovsky, 1926; Navara et al., 1994), nitrogen, phosphorus (Tennant, 1976; Brady et al., 1995), oxygen (Wiengweera and Greenway, 2004), soil hardness (Acuna et al., 2007), and microorganisms (Sivasithamparam et al., 1978). Of note is the study by Krassovsky (1926), which was the first, to our knowledge, to show differences in function related to water. Krassovsky (1926) showed that seminal roots of wheat absorbed almost 2 times the water as nodal roots per unit dry weight but that nodal roots absorbed a more diluted nutrient solution than seminal roots. Krassovsky (1926) also showed by removing seminal or nodal roots as they emerged that “seminal roots serve the main stem, while nodal roots serve the tillers” (Krassovsky, 1926). Volkmar (1997) showed, more recently, in wheat that nodal and seminal roots may sense and respond to drought differently. In millet (Pennisetum glaucum) and sorghum (Sorghum bicolor), Rostamza et al. (2013) found that millet was able to grow nodal roots in a dryer soil than sorghum, possibly because of shoot and root vigor.Open in a separate windowFigure 1.B. distachyon plant scanned at the fourth leaf stage, with the root and shoot phenotypes studied indicated. Supplemental Table S1.

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

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