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)

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

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

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

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

Analysis of the Clonal Relationship of Serotype O26:H11 Enterohemorrhagic Escherichia coli Isolates from Cattle

Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (42). In humans, infections with some STEC serotypes result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by hemolytic-uremic syndrome (HUS) (49). These STEC strains are also designated “enterohemorrhagic E. coli” (EHEC). Consequently, EHEC strains represent a subgroup of STEC with a high pathogenic potential for humans. Strains of the E. coli serogroup O26 were originally classified as enteropathogenic E. coli due to their association with outbreaks of infantile diarrhea in the 1940s. In 1977, Konowalchuk et al. (37) recognized that these bacteria produced Stx, and 10 years later, the Stx-producing E. coli O26:H11/H− strains were classified as EHEC. EHEC O26 strains constitute the most common non-O157 EHEC group associated with diarrhea and HUS in Europe (12, 21, 23, 24, 26, 27, 55, 60). Reports on an association between EHEC O26 and HUS or diarrhea from North America including the United States (15, 30, 33), South America (51, 57), Australia (22), and Asia (31, 32) provide further evidence for the worldwide spread of these organisms. Studies in Germany and Austria (26, 27) on sporadic HUS cases between 1996 and 2003 found that EHEC O26 accounted for 14% of all EHEC strains and for ∼40% of non-O157 EHEC strains obtained from these patients. A proportion of 11% EHEC O26 strains was detected in a case-control study in Germany (59) between 2001 and 2003. In the age group <3 years, the number of EHEC O26 cases was nearly equal to that of EHEC O157 cases, although the incidence of EHEC O26-associated disease is probably underestimated because of diagnostic limitations in comparison to the diagnosis of O157:H7/H− (18, 34). Moreover, EHEC O26 has spread globally (35). Beutin (6) described EHEC O26:H11/H−, among O103:H2, O111:H, O145:H28/H−, and O157:H7/H−, as the well-known pathogenic “gang of five,” and Bettelheim (5) warned that we ignore the non-O157 STEC strains at our peril.EHEC O26 strains produce Stx1, Stx2, or both (15, 63). Moreover, these strains contain the intimin-encoding eae gene (11, 63), a characteristic feature of EHEC (44). In addition, EHEC strains possess other markers associated with virulence, such as a large plasmid that carries further potential virulence genes, e.g., genes coding for EHEC hemolysin (EHEC-hlyA), a catalase-peroxidase (katP), and an extracellular serine protease (espP) (17, 52). The efa1 (E. coli factor for adherence 1) gene was identified as an intestinal colonization factor in EHEC (43). EHEC O26 represents a highly dynamic group of organisms that rapidly generate new pathogenic clones (7, 8, 63).Ruminants, especially cattle, are considered the primary reservoir for human infections with EHEC. Therefore, the aim of this study was the molecular characterization of bovine E. coli field isolates of serogroup O26 using a panel of typical virulence markers. The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O26 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.In our study, 56 bovine E. coli O26:H11 isolates and one bovine O26:H32 isolate were analyzed for EHEC virulence-associated factors. The isolates had been obtained from three different beef farms during a long-term study. They were detected in eight different cattle in farm A over a period of 15 months (detected on 10 sampling days), in 3 different animals in farm C over a period of 8 months (detected on 3 sampling days), and in one cow on one sampling day in farm D (Table ​(Table1)1) (28).

TABLE 1.

Typing of E. coli O26 isolates

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

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

Single Nucleotide Polymorphism-Based Diagnostic System for Crop-Associated Sclerotinia Species

A molecular diagnostic system using single nucleotide polymorphisms (SNPs) was developed to identify four Sclerotinia species: S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. DNAs of samples are hybridized with each of five 15-bp oligonucleotide probes containing an SNP site midsequence unique to each species. For additional verification, hybridizations were performed using diagnostic single nucleotide substitutions at a 17-bp sequence of the calmodulin locus. The accuracy of these procedures was compared to that of a restriction fragment length polymorphism (RFLP) method based on Southern hybridizations of EcoRI-digested genomic DNA probed with the ribosomal DNA-containing plasmid probe pMF2, previously shown to differentiate S. sclerotiorum, S. minor, and S. trifoliorum. The efficiency of the SNP-based assay as a diagnostic test was evaluated in a blind screening of 48 Sclerotinia isolates from agricultural and wild hosts. One isolate of Botrytis cinerea was used as a negative control. The SNP-based assay accurately identified 96% of Sclerotinia isolates and could be performed faster than RFLP profiling using pMF2. This method shows promise for accurate, high-throughput species identification.Sclerotinia is distinguished morphologically from other genera in the Sclerotiniaceae (Ascomycota, Pezizomycotina, Leotiomycetes) by the production of tuberoid sclerotia that do not incorporate host tissue, by the production of microconidia that function as spermatia but not as a disseminative asexual state, and by the development of a layer of textura globulosa composing the outer tissue of apothecia (8). Two hundred forty-six species of Sclerotinia have been reported, most distinguished morphotaxonomically (Index Fungorum [www.indexfungorum.org]). These include the four species of agricultural importance now recognized plus many that are imperfectly known, seldom collected, or apparently endemic to relatively small geographic areas (2, 5, 6, 7, 8, 9, 17).The main species of phythopathological interest in the genus Sclerotinia are S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. Sclerotinia species 1 is an important cause of disease in vegetables in Alaska (16) and has been found in association with wild Taraxacum sp., Caltha palustris, and Aconitum septentrionalis in Norway (7). It is morphologically indistinguishable from S. sclerotiorum, but it was shown to be a distinct species based on distinctive polymorphisms in sequences from internal transcribed spacer 2 (ITS2) of the nuclear ribosomal repeat (7). The other three species have been delimited using morphological, cytological, biochemical, and molecular characters (3, 8, 9, 10, 12, 15). Interestingly, given that the ITS is sufficiently polymorphic in many fungal genera to resolve species, in Sclerotinia, only species 1 and S. trifoliorum are distinguished by characteristic ITS sequence polymorphisms; S. sclerotiorum and S. minor cannot be distinguished based on ITS sequence (2, 7).Sclerotinia sclerotiorum is a necrotrophic pathogen with a broad host range (1). S. minor has a more restricted host range but causes disease in a variety of important crops such as lettuce, peanut, and sunflower crops (11). S. trifoliorum has a much narrower host range, limited to the Fabaceae (3, 8, 9). Sclerotial and ascospore characteristics also serve to differentiate among the three species. Sclerotinia minor has small sclerotia that develop throughout the colony in vitro and aggregate to form crusts on the host, while the sclerotia of S. sclerotiorum and S. trifoliorum are large and form at the colony periphery in vitro, remaining separate on the host (8, 9). The failure of an isolate to produce sclerotia or apothecia in vitro is not unusual, especially after serial cultivation (8). The presence of dimorphic, tetranucleate ascospores characterizes S. trifoliorum, while S. sclerotiorum and S. minor both have uniformly sized ascospores that are binucleate and tetranucleate, respectively (9, 14).With the apparent exception of Sclerotinia species 1, morphological characteristics are sufficient to delimit Sclerotinia species given that workers have all manifestations of the life cycle in hand. In cultures freshly isolated from infected plants, investigators usually have mycelia and sclerotia but not apothecia. Restriction fragment length polymorphisms (RFLPs) in ribosomal DNA (rDNA) are diagnostic for Sclerotinia species (3, 10), but the assay requires cloned probes (usually accessed from other laboratories) hybridized to Southern blots from vertical gels, an impractical procedure for large samples. We have analyzed sequence data from previous phylogenetic studies (2) and have identified diagnostic variation for the rapid identification of the four Sclerotinia species. The single nucleotide polymorphism (SNP) assay that we report here is amenable to a high throughput of samples and requires only PCR amplification with a standard set of primers and oligonucleotide hybridizations to Southern blots in a dot format.The SNP assay was performed using two independent sets of species-specific oligonucleotide probes, all with SNP sites shown to differentiate the four Sclerotinia species (Fig. ​(Fig.1).1). A panel of 49 anonymously coded isolates (Table ​(Table1)1) was screened using these species-specific SNP probes, as outlined in Fig. ​Fig.1.1. The assay was validated by comparison to Southern hybridizations of EcoRI-digested genomic DNA hybridized with pMF2, a plasmid probe containing the portion of the rDNA repeat with the 18S, 5.8S, and 26S rRNA cistrons of Neurospora crassa (4, 10).Open in a separate windowFIG. 1.Protocol for the SNP-based identification of Sclerotinia species, with diagnostic SNP sites underlined and in boldface type for each hybridization probe.

TABLE 1.

Isolates and hybridization results for all SNP-based oligonucleotide probes^f

Growth of Arthrobacter sp. Strain JBH1 on Nitroglycerin as the Sole Source of Carbon and Nitrogen

Arthrobacter sp. strain JBH1 was isolated from nitroglycerin-contaminated soil by selective enrichment. Detection of transient intermediates and simultaneous adaptation studies with potential intermediates indicated that the degradation pathway involves the conversion of nitroglycerin to glycerol via 1,2-dinitroglycerin and 1-mononitroglycerin, with concomitant release of nitrite. Glycerol then serves as the source of carbon and energy.Nitroglycerin (NG) is manufactured widely for use as an explosive and a pharmaceutical vasodilator. It has been found as a contaminant in soil and groundwater (7, 9). Due to NG''s health effects as well as its highly explosive nature, NG contamination in soils and groundwater poses a concern that requires remedial action (3). Natural attenuation and in situ bioremediation have been used for remediation in soils contaminated with certain other explosives (16), but the mineralization of NG in soil and groundwater has not been reported.To date, no pure cultures able to grow on NG as the sole carbon, energy, and nitrogen source have been isolated. Accashian et al. (1) observed growth associated with the degradation of NG under aerobic conditions by a mixed culture originating from activated sludge. The use of NG as a source of nitrogen has been studied in mixed and pure cultures during growth on alternative sources of carbon and energy (3, 9, 11, 20). Under such conditions, NG undergoes a sequential denitration pathway in which NG is transformed to 1,2-dinitroglycerin (1,2DNG) or 1,3DNG followed by 1-mononitroglycerin (1MNG) or 2MNG and then glycerol, under both aerobic and anaerobic conditions (3, 6, 9, 11, 20), and the enzymes involved in denitration have been characterized in some detail (4, 8, 15, 21). Pure cultures capable of completely denitrating NG as a source of nitrogen when provided additional sources of carbon include Bacillus thuringiensis/cereus and Enterobacter agglomerans (11) and a Rhodococcus species (8, 9). Cultures capable of incomplete denitration to MNG in the presence of additional carbon sources were identified as Pseudomonas putida, Pseudomonas fluorescens (4), an Arthobacter species, a Klebsiella species (8, 9), and Agrobacterium radiobacter (20).Here we describe the isolation of bacteria able to degrade NG as the sole source of carbon, nitrogen, and energy. The inoculum for selective enrichment was soil historically contaminated with NG obtained at a facility that formerly manufactured explosives located in the northeastern United States. The enrichment medium consisted of minimal medium prepared as previously described (17) supplemented with NG (0.26 mM), which was synthesized as previously described (18). During enrichment, samples of the inoculum (optical density at 600 nm [OD₆₀₀] ∼ 0.03) were diluted 1/16 in fresh enrichment medium every 2 to 3 weeks. Isolates were obtained by dilution to extinction in NG-supplemented minimal medium. Cultures were grown under aerobic conditions in minimal medium at pH 7.2 and 23°C or in tryptic soy agar (TSA; 1/4 strength).Early stages of enrichment cultures required extended incubation with lag phases of over 200 h and exhibited slow degradation of NG (less than 1 μmol substrate/mg protein/h). After a number of transfers over 8 months, the degradation rates increased substantially (2.2 μmol substrate/mg protein/h). A pure culture capable of growth on NG was identified based on 16S rRNA gene analysis (504 bp) as an Arthrobacter species with 99.5% similarity to Arthrobacter pascens (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU246730","term_id":"289474572","term_text":"GU246730"}}GU246730). Purity of the cultures was confirmed microscopically and by formation of a single colony type on TSA plates. 16S gene sequencing and identification were done by MIDI Labs (Newark, DE) and SeqWright DNA Technology Services (Houston, TX). The Arthrobacter cells stained primarily as Gram-negative rods with a small number of Gram-positive cocci (data not shown); Gram variability is also a characteristic of the closely related Arthrobacter globiformis (2, 19). The optimum growth temperature is 30°C, and the optimum pH is 7.2. Higher pH values were not investigated because NG begins to undergo hydrolysis above pH 7.5 (data not shown). The isolated culture can grow on glycerol, acetate, succinate, citrate, and lactate, with nitrite as the nitrogen source. Previous authors described an Arthrobacter species able to use NG as a nitrogen source in the presence of additional sources of carbon. However, only dinitroesters were formed, and complete mineralization was not achieved (9).To determine the degradation pathway, cultures of the isolated strain (5 ml of inoculum grown on NG to an OD₆₀₀ of 0.3) were grown in minimal medium (100 ml) supplemented with NG at a final concentration of 0.27 mM. Inoculated bottles and abiotic controls were continuously mixed, and NG, 1,2DNG, 1,3DNG, 1MNG, 2MNG, nitrite, nitrate, CO₂, total protein, and optical density were measured at appropriate intervals. Nitroesters were analyzed with an Agilent high-performance liquid chromatograph (HPLC) equipped with an LC-18 column (250 by 4.6 mm, 5 μm; Supelco) and a UV detector at a wavelength of 214 nm (13). Methanol-water (50%, vol/vol) was used as the mobile phase at a flow rate of 1 ml/min. Nitrite and nitrate were analyzed with an ion chromatograph (IC) equipped with an IonPac AS14A anion-exchange column (Dionex, CA) at a flow rate of 1 ml/min. Carbon dioxide production was measured with a Micro Oxymax respirometer (Columbus Instruments, OH), and total protein was quantified using the Micro BCA protein assay kit (Pierce Biotechnology, IL) according to manufacturer''s instructions. During the degradation of NG the 1,2DNG concentration was relatively high at 46 and 72 h (Fig. ​(Fig.1).1). 1,3DNG, detected only at time zero, resulted from trace impurities in the NG stock solution. Trace amounts of 1MNG appeared transiently, and trace amounts of 2MNG accumulated and did not disappear. Traces of nitrite at time zero were from the inoculum. The concentration of NG in the abiotic control did not change during the experiment (data not shown).Open in a separate windowFIG. 1.Growth of strain JBH1 on NG. ×, NG; ▵, 1,2DNG; ⋄, 1MNG; □, 2MNG; ○, protein.Results from the experiment described above were used to calculate nitrogen and carbon mass balances (Tables ​(Tables11 and ​and2).2). Nitrogen content in protein was approximated using the formula C₅H₇O₂N (14). Because all nitrogen was accounted for throughout, we conclude that the only nitrogen-containing intermediate compounds are 1,2DNG and 1MNG, which is consistent with previous studies (6, 9, 20). The fact that most of the nitrogen was released as nitrite is consistent with previous reports of denitration catalyzed by reductase enzymes (4, 8, 21). The minor amounts of nitrate observed could be from abiotic hydrolysis (5, 12) or from oxidation of nitrite. Cultures supplemented with glycerol or other carbon sources assimilated all of the nitrite (data not shown).

TABLE 1.

Nitrogen mass balance

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 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