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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.
Open in a separate windowaThe annotated genome for S. sclerotiorum strain 1980 (ATCC 18683) is publicly available through the Broad Institute, Cambridge, MA (http://www.broad.mit.edu/annotation/genome/sclerotinia_sclerotiorum/Home.html).bAll isolates from New York were provided by Gary C. Bergstrom, Cornell University, Ithaca, NY. Isolates Ss001 and Ssp005 were submitted as S. sclerotiorum, and Ssp001 through Ssp004 were submitted as S. trifoliorum.cAll isolates from Alaska, submitted as Sclerotinia species 1, were provided by Lori Winton, USDA-ARS Subarctic Agricultural Research Unit, University of Alaska, Fairbanks.dAll isolates from Finland, submitted as S. trifoliorum, were provided by Tapani Yli-Mattila, University of Turku, Turku, Finland.eAll isolates from Australia, presumed to be S. sclerotiorum but requiring species confirmation, were provided by Martin Barbetti, DAF Plant Protection Branch, South Perth, Australia.fThe probes that are diagnostic for S. minor, S. sclerotiorum, S. trifoliorum, and Sclerotinia species 1 are listed, with a “+” indicating a positive hybridization for the probe and a “−” indicating no hybridization of the probe. 相似文献
TABLE 1.
Isolates and hybridization results for all SNP-based oligonucleotide probesfCollector''s isolate | Anonymous code | Prescreened presumed species identity | Origin | Host | Species-specific SNP
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IGS50 | CAL448 S.trifol | CAL124 | CAL448 S.minor | RAS148 | CAL446 S.sp1 | CAL19A | CAL19B | CAL448 S.sclero | |||||
LMK18 | 49 | Botrytis cinerea | Ontario, Canada | Allium cepa | − | − | − | − | − | − | − | − | − |
FA2-1 | 3 | Sclerotinia minor | North Carolina | Arachis hypogaea | − | − | + | + | − | − | − | − | − |
W1 | 5 | Sclerotinia minor | North Carolina | Cyperus esculentus | − | − | + | + | − | − | − | − | − |
W10 | 30 | Sclerotinia minor | North Carolina | Oenothra laciniata | − | − | + | + | − | − | − | − | − |
PF1-1 | 38 | Sclerotinia minor | North Carolina | Arachis hypogaea | − | − | + | + | − | − | − | − | − |
PF18-497 | 14 | Sclerotinia minor | Oklahoma | Arachis hypogaea | − | − | + | + | − | − | − | − | − |
PF17-482 | 46 | Sclerotinia minor | Oklahoma | Arachis hypogaea | − | − | + | + | − | − | − | − | − |
PF19-519 | 48 | Sclerotinia minor | Oklahoma | Arachis hypogaea | − | − | + | + | − | − | − | − | − |
LF-27 | 20 | Sclerotinia minor | United States | Lactuca sativa | − | − | + | + | − | − | − | − | − |
AR1281 | 1 | Sclerotinia sclerotiorum | Argentina | Arachis hypogaea | − | − | − | − | − | − | + | − | + |
AR1282 | 16 | Sclerotinia sclerotiorum | Argentina | Arachis hypogaea | − | − | − | − | − | − | + | − | + |
LMK211 | 6 | Sclerotinia sclerotiorum | Canada | Brassica napus | − | − | − | − | − | − | + | − | + |
LMK57 | 25 | Sclerotinia sclerotiorum | Norway | Ranunculus ficaria | − | − | − | − | − | − | + | − | + |
LMK754 | 15 | Sclerotinia sclerotiorum | Norway | Ranunculus ficaria | − | − | − | − | − | − | − | + | + |
UR19 | 39 | Sclerotinia sclerotiorum | Uruguay | Lactuca sativa | − | − | − | − | − | − | + | − | + |
UR478 | 9 | Sclerotinia sclerotiorum | Uruguay | Lactuca sativa | − | − | − | − | − | − | + | − | + |
CA901 | 32 | Sclerotinia sclerotiorum | California | Lactuca sativa | − | − | − | − | − | − | + | − | + |
CA995 | 40 | Sclerotinia sclerotiorum | California | Lactuca sativa | − | − | − | − | − | − | + | − | + |
CA1044 | 41 | Sclerotinia sclerotiorum | California | Lactuca sativa | − | − | − | − | − | − | + | − | + |
1980a | 34 | Sclerotinia sclerotiorum | Nebraska | Phaseolus vulgaris | − | − | − | − | − | − | + | − | + |
Ss001 | 13 | Sclerotinia sclerotiorum | New Yorkb | Glycine max | − | − | − | − | − | − | + | − | + |
Ssp005 | 31 | Sclerotinia sclerotiorum | New York | Glycine max | − | − | − | − | − | − | + | − | + |
H02-V28 | 33 | Sclerotinia species 1 | Alaskac | Unknown vegetable crop | − | − | − | − | + | + | − | − | − |
H01-V14 | 26 | Sclerotinia species 1 | Alaska | Unknown vegetable crop | − | − | − | − | + | + | − | − | − |
LMK745 | 21 | Sclerotinia species 1 | Norway | Taraxacum sp. | − | − | − | − | + | + | − | − | − |
02-26 | 11 | Sclerotinia trifoliorum | Finlandd | Trifolium pratense | + | − | − | − | − | − | − | − | − |
06-14 | 29 | Sclerotinia trifoliorum | Finland | Trifolium pratense | + | + | − | − | − | − | − | − | − |
202 | 2 | Sclerotinia trifoliorum | Finland | Trifolium pratense | + | + | − | − | − | − | − | − | − |
2-L9 | 45 | Sclerotinia trifoliorum | Finland | Trifolium pratense | + | + | − | − | − | − | − | − | − |
3-A5 | 24 | Sclerotinia trifoliorum | Finland | Trifolium pratense | − | − | − | − | − | − | − | − | − |
5-L9 | 12 | Sclerotinia trifoliorum | Finland | Trifolium pratense | + | + | − | − | − | − | − | − | − |
K1 | 4 | Sclerotinia trifoliorum | Finland | Trifolium pratense | + | + | − | − | − | − | − | − | − |
K2 | 37 | Sclerotinia trifoliorum | Finland | Trifolium pratense | + | + | − | − | − | − | − | − | − |
L-112 | 23 | Sclerotinia trifoliorum | Finland | Trifolium pratense | + | + | − | − | − | − | − | − | − |
L-119 | 44 | Sclerotinia trifoliorum | Finland | Trifolium pratense | + | + | − | − | − | − | − | − | − |
LMK36 | 19 | Sclerotinia trifoliorum | Tasmania | Trifolium repens | + | + | − | − | − | − | − | − | − |
Ssp001 | 18 | Sclerotinia trifoliorum | New York | Lotus corniculatus | + | + | − | − | − | − | − | − | − |
Ssp002 | 10 | Sclerotinia trifoliorum | New York | Lotus corniculatus | + | + | − | − | − | − | − | − | − |
Ssp003 | 28 | Sclerotinia trifoliorum | New York | Lotus corniculatus | + | + | − | − | − | − | − | − | − |
Ssp004 | 36 | Sclerotinia trifoliorum | New York | Lotus corniculatus | + | + | − | − | − | − | − | − | − |
LMK47 | 43 | Sclerotinia trifoliorum | Virginia | Medicago sativa | + | + | − | − | − | − | − | − | − |
MBRS-1 | 27 | Unknown | Australiae | Brassica spp. | − | − | − | − | − | − | + | − | + |
MBRS-2 | 7 | Unknown | Australia | Brassica spp. | − | − | − | − | − | − | + | − | + |
MBRS-3 | 42 | Unknown | Australia | Brassica spp. | − | − | − | − | − | − | + | − | + |
MBRS-5 | 22 | Unknown | Australia | Brassica spp. | − | − | − | − | − | − | + | − | + |
WW-1 | 35 | Unknown | Australia | Brassica spp. | − | − | − | − | − | − | + | − | + |
WW-2 | 8 | Unknown | Australia | Brassica spp. | − | − | − | − | − | − | + | − | + |
WW-3 | 17 | Unknown | Australia | Brassica spp. | − | − | − | − | − | − | + | − | + |
WW-4 | 47 | Unknown | Australia | Brassica spp. | − | − | − | − | − | − | + | − | + |
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Lutz Geue Sabrina Klare Christina Schnick Birgit Mintel Katharina Meyer Franz J. Conraths 《Applied and environmental microbiology》2009,75(21):6947-6953
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).
Open in a separate windowastx1/stx2, gene stx1 or stx2.befa1 was detected by two hybridizations (with lifA1-lifA2 and lifA3-lifA4 probes). +/+, complete gene; +/− or −/+, incomplete gene; −/−, efa1 negative.The serotyping of the O26 isolates was confirmed by the results of the fliC PCR-restriction fragment length polymorphism (RFLP) analysis performed according to Fields et al. (25), with slight modifications described by Zhang et al. (62). All O26:H11 isolates showed the H11 pattern described by Zhang et al. (62). In contrast, the O26:H32 isolate demonstrated a different fliC RFLP pattern that was identical to the H32 pattern described by the same authors. It has been demonstrated that EHEC O26:H11 strains belong to at least four different sequence types (STs) in the common clone complex 29 (39). In the multilocus sequence typing analysis for E. coli (61), the tested five EHEC O26:H11 isolates (WH-01/02/003-1, WH-01/20/005-3, WH-01/27/009-9, WH-03/14/004-8, and WH-04/22/001-1) of different farms and clusters were characterized as two sequence types (ST 21 and ST 396). The isolates from farms A and C belong to ST 21, the most frequent ST of EHEC O26:H11 isolates found in humans and animals (39), but the single isolate from farm D was characterized as ST 396.Typing and subtyping of genes (stx1 and/or stx2, eae, tir, espA, espB, EHEC-hlyA, katP, and espP) associated with EHEC were performed with LightCycler fluorescence PCR (48) and different block-cycler PCRs. To identify the subtypes of the stx2 genes and of the locus of enterocyte effacement-encoding genes eae, tir, espA, and espB, the PCR products were digested by different restriction endonucleases (19, 26, 46). The complete pattern of virulence markers was detected in most bovine isolates examined in our study. An stx1 gene was present in all O26 isolates. In addition, an stx2 gene was found in nine O26:H11 isolates in farm A and in three isolates of the same type in farm C, as well as in the O26:H32 isolate. Both Stx1 and Stx2 were closely related to families of Stx1 and Stx2 variants or alleles. EHEC isolates with stx2 genes are significantly more often associated with HUS and other severe disease manifestations than isolates with an stx1 gene, which are more frequently associated with uncomplicated diarrhea and healthy individuals (13). In contrast to STEC strains harboring stx2 gene variants, however, STEC strains of the stx2 genotype were statistically significantly associated with HUS (26). The stx2 genotype was found in all O26 isolates with an stx2 gene, while the GK3/GK4 amplification products after digestion with HaeIII and FokI restriction enzymes showed the typical pattern for this genotype described by Friedrich et al. (26). The nucleotide sequences of the A and B subunits of the stx2 gene of the selected bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. ) were identical to the stx2 genes of different sorbitol-fermenting EHEC O157:H− strains associated with human HUS cases and other EHEC infections in Germany ( EU70049110) and 99.3% identical in their DNA sequences to the stx2 gene of the EHEC type strain EDL933, a typical O157:H7 isolate from an HUS patient. A characteristic stx1 genotype was present in all O26 isolates. The nucleotide sequences of the A and B subunits of the stx1 gene of the tested bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. ) were nearly identical to those of the stx1 genes of the EHEC O26:H11 reference type strains H19 and DEC10B, which had been associated with human disease outbreaks in Canada and Australia. Nucleotide exchanges typical for stx1c and stx1d subtypes as described by Kuczius et al. ( EU70049038) were not found. All bovine O26:H11 strains produced an Stx1 with high cytotoxicity for Vero cells tested by Stx enzyme-linked immunosorbent assay and Vero cell neutralization assay (53). The Stx2 cytotoxicity for Vero cells was also very high in the O26:H11 isolates.Not only factors influencing the basic and inducible Stx production are important in STEC pathogenesis. It has been suggested that the eae and EHEC-hlyA genes are likely contributors to STEC pathogenicity (2, 3, 13, 50). Ritchie et al. (50) found both genes in all analyzed HUS-associated STEC isolates. In all O26:H11 isolates we obtained, stx genes were present in combination with eae genes. Only the O26:H32 isolate lacked an eae gene. To date, 10 distinct variants of eae have been described (1, 19, 36, 45, 47). Some serotypes were closely associated with a particular intimin variant: the O157 serogroup was linked to γ-eae, the O26 serogroup to β-eae, and the O103 serogroup to ɛ-eae (4, 19, 20, 58). Our study confirms these associations. All bovine O26:H11 isolates were also typed as members of the β-eae subgroup. A translocated intimin receptor gene (tir gene) and the type III secreted proteins encoded by the espA and espB genes were found in all 56 O26:H11 isolates but not in the O26:H32 isolate. These other tested locus of enterocyte effacement-associated genes belonged to the β-subgroups. These results are in accord with the results of China et al. (19), who detected the pathotypes β-eae, β-tir, β-espA, and β-espB in all investigated human O26 strains. Like the eae gene, the EHEC-hlyA gene was found in association with severe clinical disease in humans (52). Aldick et al. (2) showed that EHEC hemolysin is toxic (cytolytic) to human microvascular endothelial cells and may thus contribute to the pathogenesis of HUS. In our study, the EHEC-hlyA gene was detected in 50 of the 56 bovine E. coli O26:H11 isolates which harbored virulence-associated plasmids of different sizes (Table (Table1).1). The presence of virulence-associated plasmids corresponded to the occurrence of additional virulence markers such as the espP and katP genes (17). The katP gene and the espP gene were detected in 49 and 50 of the 56 O26:H11 isolates, respectively. The espP gene was missing in six of the seven bovine O26:H11 isolates in which the katP genes were also absent. Both genes were not found in the O26:H32 isolate (Table (Table1).1). Although we found large plasmids of the same size in O26:H11 isolates, they lacked one or more of the plasmid-associated virulence factors (Table (Table1).1). Two DNA probes were used to detect the efa1 genes by colony hybridization. (DNA probes were labeled with digoxigenin [DIG] with lifA1-lifA2 and lifA3-lifA4 primers [14] using the PCR DIG probe synthesis kit [Roche Diagnostics, Mannheim, Germany]; DIG Easy Hyb solution [Roche] was used for prehybridization and hybridization.) Positive results with both DNA probes were obtained for 52 of 56 E. coli O26:H11 isolates. A positive signal was only found in three isolates with the lifA1-lifA2 DNA probe and in one isolate with the lifA3-lifA4 probe. An efa1 gene was not detected in the O26:H32 isolate (Table (Table11).We also analyzed the spatial and temporal behavior of the O26:H11/H32 isolates in the beef herds by cluster analysis (conducted in PAUP* for Windows version 4.0, 2008 [http://paup.csit.fsu.edu/about.html]). This was performed with distance matrices using the neighbor-joining algorithm, an agglomerative cluster method which generates a phylogenetic tree. The distance matrices were calculated by pairwise comparisons of the fragmentation patterns produced by genomic typing through pulsed-field gel electrophoresis analysis with four restriction endonucleases (XbaI, NotI, BlnI, and SpeI) and the presence or absence of potential virulence markers (Fig. (Fig.11 and Table Table1).1). To this end, the total character difference was used, which counts the pairwise differences between two given patterns. During a monitoring program of 3 years in four cattle farms (29), different O26:H11 cluster groups and one O26:H32 isolate were detected in three different farms. The genetic distance of the O26:H32 isolate was very high relative to the O26:H11 isolates. Therefore, the O26:H32 isolate was outgrouped. The O26:H11 isolates of each farm represented independent cluster groups. The single isolate from farm D fitted better to the isolates from farm C than to those from farm A. This finding is in accord with the geographical distance between the farms. The fact that the farms were located in neighboring villages may suggest that direct or indirect connections between the farms were possible (e.g., by person contacts or animal trade). However, the isolates from farm C and farm D belonged to different sequence types (ST 21 and ST 396), which may argue against a direct connection. Interestingly, O26:H11 isolates with and without stx2 genes were detected in the same clusters. This phenomenon was observed in both farm A and farm C. In farm A, the isolates with additional stx2 genes were found in animal 27 and were grouped in clusters 8 and 9 (day 218). An stx2 gene was repeatedly found (four isolates) in the same animal (animal 27). The isolates grouped in cluster 8 on a later day of sampling (day 478). All other O26:H11 isolates grouped in the same clusters and obtained from the same animals (27 and 29) on different sampling days lacked an stx2 gene. Also, the isolates obtained from animal 27 on previous sampling days, which grouped in clusters 3 and 5, exhibited no stx2 genes. In farm C, the three isolates with additional stx2 genes obtained from animal 7 grouped in clusters 11 and 12. An stx2 gene was absent from all other O26:H11 isolates grouped in the same cluster 12 on later sampling days, and no other isolates of cluster 11 were found later on. However, we detected members of many clusters over relatively long periods (clusters 5, 8, and 9 in farm A and cluster 12 in farm C), but members of other clusters were only found on single occasions. This patchy temporal pattern is apparently not a unique property of O26:H11, as we found similar results for cluster groups of other EHEC serotypes of bovine origin (28). The isolates grouped in the dominant cluster 8 were found on 5 of 9 sampling days over a period of 10 months. In contrast, we found the members of clusters 4, 5, 9, and 12 only on two nonconsecutive sampling days. The period during which isolates of these groups were not detected was particularly long for cluster 4 (231 days). We also observed the coexistence of different clusters over long periods in the same farm and in the same cattle (clusters 8 and 9), while one of the clusters dominated. Transmission of clusters between cattle was also observed. These results suggest that some of the EHEC O26:H11 strains had the potential for a longer persistence in the host population, while others had not. The reasons for this difference are not yet clear. Perhaps the incomplete efa1 gene found in isolates of clusters which were only detected once might explain why some strains disappeared rapidly. Efa1 has been discussed as a potential E. coli colonization factor for the bovine intestine used by non-O157 STEC, including O26 (54, 56). The O165:H25 cluster detected during a longer period in farm B may have disappeared after it had lost its efa1 gene (28). The precise biological activity of Efa1 in EHEC O26 is not yet known, but it has been demonstrated that the molecule is a non-Stx virulence determinant which can increase the virulence of EHEC O26 in humans (8).Open in a separate windowFIG. 1.Neighbor-joining tree of bovine E. coli O26:H11/H32 strains based on the restriction pattern obtained after digestion with XbaI, NotI, BlnI, and SpeI.We distinguished 12 different clusters, but complete genetic identity was only found in two isolates. The variations in the O26:H11 clusters may be due to increasing competition between the bacterial populations of the various subtypes in the bovine intestine or to potential interactions between EHEC O26:H11 and the host.The ephemeral occurrence of additional stx2 genes in different clusters and farms may be the result of recombination events due to horizontal gene transfer (16). The loss of stx genes may occur rapidly in the course of an infection, but the reincorporation by induction of an stx-carrying bacteriophage into the O26:H11 strains is possible at any time (9, 40). Nevertheless, an additional stx2 gene may increase the dangerousness of the respective EHEC O26:H11 strains. While all patients involved in an outbreak caused by an EHEC O26:H11 strain harboring the gene encoding Stx2 developed HUS (41), the persons affected by another outbreak caused by an EHEC O26:H11 strain that produced exclusively Stx1 had only uncomplicated diarrhea (60).In conclusion, our results showed that bovine O26:H11 isolates can carry virulence factors of EHEC that are strongly associated with EHEC-related disease in humans, particularly with severe clinical manifestations such as hemorrhagic colitis and HUS. Therefore, strains of bovine origin may represent a considerable risk for human infection. Moreover, some clusters of EHEC O26:H11 persisted in cattle and farms over longer periods, which may increase the risk of transmission to other animals and humans even further. 相似文献
TABLE 1.
Typing of E. coli O26 isolatesSampling day, source, and isolate | Serotype | Virulence profile by: | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
fliC PCR-RFLP | stx1 gene | stx2 gene | Stx1 (toxin) | Stx2 (toxin) | Subtype(s) | efa1 geneb | EHEC-hlyA gene | katP gene | espP gene | Plasmid size(s) in kb | Cluster | ||||||
stx1/stx2 | eae | tir | espA | espB | |||||||||||||
Day 15 | |||||||||||||||||
Animal 6 (farm A) | |||||||||||||||||
WH-01/06/002-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
WH-01/06/002-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
WH-01/06/002-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
Animal 8 (farm A) | |||||||||||||||||
WH-01/08/002-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
Animal 26 (farm A) | |||||||||||||||||
WH-01/26/001-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 7 |
WH-01/26/001-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
WH-01/26/001-6 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
WH-01/26/001-7 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/− | + | + | + | 110, 12 | 7 |
Day 29 | |||||||||||||||||
Animal 2 (farm A) | |||||||||||||||||
WH-01/02/003-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-6 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | + | + | 110, 12 | 6 |
WH-01/02/003-7 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-8 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | −/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-9 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110 | 6 |
WH-01/02/003-10 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110 | 6 |
Animal 26 (farm A) | |||||||||||||||||
WH-01/26/002-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 5 |
WH-01/26/002-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 5 |
WH-01/26/002-8 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 5 |
WH-01/26/002-9 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | − | − | 110, 12 | 5 |
WH-01/26/002-10 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 5 |
Day 64 | |||||||||||||||||
Animal 20 (farm A) | |||||||||||||||||
WH-01/20/005-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | − | − | 130, 2.5 | 2 |
Day 78 | |||||||||||||||||
Animal 29 (farm A) | |||||||||||||||||
WH-01/29/002-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/− | + | − | − | 130, 12, 2.5 | 4 |
WH-01/29/002-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 4 |
WH-01/29/002-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 4 |
WH-01/29/002-4 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 4 |
WH-01/29/002-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | − | + | 130, 12, 2.5 | 4 |
Day 106 | |||||||||||||||||
Animal 27 (farm A) | |||||||||||||||||
WH-01/27/005-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/− | + | + | + | 145, 110, 12 | 3 |
WH-01/27/005-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 5 |
WH-01/27/005-6 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | − | − | 130, 12, 2.5 | 5 |
Day 113 | |||||||||||||||||
Animal 7 (farm C) | |||||||||||||||||
WH-04/07/001-2 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | − | + | + | 55, 35, 2.5 | 11 |
WH-04/07/001-4 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 55 | 12 |
WH-04/07/001-6 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 55 | 12 |
Day 170 | |||||||||||||||||
Animal 22 (farm C) | |||||||||||||||||
WH-04/22/001-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 6.3 | 12 |
WH-04/22/001-4 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 6.3 | 12 |
WH-04/22/001-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 6.3 | 12 |
Day 176 | |||||||||||||||||
Animal 14 (farm D) | |||||||||||||||||
WH-03/14/004-8 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | + | + | 110 | 10 |
Day 218 | |||||||||||||||||
Animal 27 (farm A) | |||||||||||||||||
WH-01/27/009-1 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
WH-01/27/009-2 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
WH-01/27/009-3 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 8 |
WH-01/27/009-8 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | − | − | 110, 12 | 8 |
WH-01/27/009-9 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
Day 309 | |||||||||||||||||
Animal 29 (farm A) | |||||||||||||||||
WH-01/29/010-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 35, 12 | 4 |
WH-01/29/010-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | − | − | 130, 55, 35 | 8 |
WH-01/29/010-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 35, 12 | 8 |
Day 365 | |||||||||||||||||
Animal 8 (farm C) | |||||||||||||||||
WH-04/08/008-6 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 55 | 12 |
Day 379 | |||||||||||||||||
Animal 9 (farm A) | |||||||||||||||||
WH-01/09/016-2 | O26:H32 | H32 | + | + | − | − | stx1/stx2 | − | − | − | − | −/− | − | − | − | 145, 130, 1.8 | 1 |
Animal 27 (farm A) | |||||||||||||||||
WH-01/27/014-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
WH-01/27/014-4 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
WH-01/27/014-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 8 |
Day 407 | |||||||||||||||||
Animal 29 (farm A) | |||||||||||||||||
WH-01/29/013-4 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 2.5 | 8 |
WH-01/29/013-7 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 2.5 | 8 |
Day 478 | |||||||||||||||||
Animal 27 (farm A) | |||||||||||||||||
WH-01/27/017-1 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 8 |
WH-01/27/017-5 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 8 |
WH-01/27/017-6 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110 | 8 |
WH-01/27/017-7 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110 | 8 |
WH-01/27/017-10 | O26:H11 | H11 | + | − | + | + | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 8 |
5.
6.
Camilla L. Nesb? Rajkumari Kumaraswamy Marlena Dlutek W. Ford Doolittle Julia Foght 《Applied and environmental microbiology》2010,76(14):4896-4900
All cultivated Thermotogales are thermophiles or hyperthermophiles. However, optimized 16S rRNA primers successfully amplified Thermotogales sequences from temperate hydrocarbon-impacted sites, mesothermic oil reservoirs, and enrichment cultures incubated at <46°C. We conclude that distinct Thermotogales lineages commonly inhabit low-temperature environments but may be underreported, likely due to “universal” 16S rRNA gene primer bias.Thermotogales, a bacterial group in which all cultivated members are anaerobic thermophiles or hyperthermophiles (5), are rarely detected in anoxic mesothermic environments, yet their presence in corresponding enrichment cultures, bioreactors, and fermentors has been observed using metagenomic methods and 16S rRNA gene amplification (6) (see Table S1 in the supplemental material). The most commonly detected lineage is informally designated here “mesotoga M1” (see Table S1 in the supplemental material). PCR experiments indicated that mesotoga M1 sequences amplified inconsistently using “universal” 16S rRNA gene primers, perhaps explaining their poor detection in DNA isolated from environmental samples (see text and Table S2 in the supplemental material). We therefore designed three 16S rRNA PCR primer sets (Table (Table1)1) targeting mesotoga M1 bacteria and their closest cultivated relative, Kosmotoga olearia. Primer set A was the most successful set, detecting a wider diversity of Thermotogales sequences than set B and being more Thermotogales-specific than primer set C (Table (Table22).
Open in a separate windowaHeterogeneity hot spots identified in reference 1.
Open in a separate windowaSee the supplemental material for site and methodological details. NA, not applicable; ND, not determined.bThe number of OTUs observed at a 0.01 distance cutoff is given for each primer set. The numbers of clones with Thermotogales sequences are in parentheses. —, PCR was attempted but no Thermotogales sequences were obtained or the PCR consistently failed.c+, sequence(s) detected; −, not detected. For more information on the enrichments, see the text and Table S3 in the supplemental material.dFrom April to May 2004, the temperature at the depth where the sample was taken was 12°C (7).eThere were no water samples from DWH and HSAT available for enrichment cultures, and no DNA was available from HWH.fThis reservoir has been treated with biocides; moreover, at this site, the water is filtered before being reinjected into the reservoir.gTemperatures of the oil pool where the water sample was obtained. The HSAT facility receives water from two oil pools, one at 41°C and one at 50°C.hWe screened DNA from samples taken in 2006 and 2008 but detected the same sequences in both, so sequences from the two samples were pooled.iThe mesotoga M1 and Kosmotoga sequences from DWH and DF were >99% similar and were assembled into one sequence in Fig. Fig.11.jThis reservoir has been injected with water from a neighboring oil reservoir.Since the putative mesophilic Thermotogales have been overwhelmingly associated with polluted and hydrocarbon-impacted environments and mesothermic oil reservoirs are the only natural environments where mesotoga M1 sequences previously were detected (see Table S1 in the supplemental material), we selected four oil reservoirs with in situ temperatures of 14°C to 53°C and two temperate, chronically hydrocarbon-impacted sites for analysis (Table (Table2).2). Total community DNA was extracted, the 16S rRNA genes were amplified, cloned, and sequenced as described in the supplemental material. 相似文献
TABLE 1.
Primers targeting mesotoga M1 bacteria constructed and used in this studyPrimer | Sequence (5′ to 3′) | Position in mesotoga 16S rRNA gene | No. of heterogeneity hot spotsa | Potential primer match in other Thermotogales lineages |
---|---|---|---|---|
Primer set A | 1 (helix 17) | |||
NMes16S.286F | CGGCCACAAGGAYACTGAGA | 286 | Perfect match in Kosmotoga olearia. The last 7 or 8 nucleotides at the 3′ end are conserved in other Thermotogales lineages. | |
NMes16S.786R | TGAACATCGTTTAGGGCCAG | 786 | One 5′ mismatch in Kosmotoga olearia and Petrotoga mobilis; 2-4 internal and 5′ mismatches in other lineages | |
Primer set B | None | |||
BaltD.42F | ATCACTGGGCGTAAAGGGAG | 540 | Perfect match in Kosmotoga olearia; one or two 3′ mismatches in most other Thermotogales lineages | |
BaltD.494R | GTGGTCGTTCCTCTTTCAAT | 992 | No match in other Thermotogaleslineages. The primer is located in heterogeneity hot spot helices 33 and 34. This primer also fails to amplify some mesotoga M1 sequences. | |
Primer set C | 9 (all 9 regions) | |||
TSSU-3F | TATGGAGGGTTTGATCCTGG | 3 | Perfect match in Thermotoga spp., Kosmotoga olearia, and Petrotoga mobilis; two or three 5′ mismatches in other Thermotogales lineages; one 5′ mismatch to mesotoga M1 16S rRNA genes | |
Mes16S.R | ACCAACTCGGGTGGCTTGAC | 1390 | One 5′ mismatch in Kosmotoga olearia; 1-3 internal or 5′ mismatches in other Thermotogales lineages |
TABLE 2.
Mesotoga clade sequences detected in environmental samples and enrichment cultures screened in this studyaSite (abbreviation) | Temp in situ(°C) | Waterflooded | Environmental samplesb | Enrichment cultures | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Primer set A | Primer set B | Primer set C | Thermotogalesdetected by primer setc: | Lineage(s) detected | ||||||||
No. of OTUs (no. of clones) | Lineage | No. of OTUs (no. of clones) | Lineage | No. of OTUs (no. of clones) | Lineage | A | B | C | ||||
Sidney Tar Ponds sediment (TAR) | Temperate | NA | 1 (5) | M1 | 1 | M1 | — | — | + | + | + | M1, M2, M5 |
Oil sands settling basin tailings (05mlsb) | ∼12d | NA | — | — | 1 (6) | M1 | — | — | − | + | − | M1 |
Grosmont A produced water (GrosA) | 20 | No | 1 (15) | M1 | 1 (22) | M1 | 2 (14) | M1 | + | + | + | M1 |
Foster Creek produced water (FC) | 14 | No | 1 (21) | M1 | 1 (23) | M1 | 1 (1) | M1 | + | ND | − | M1 |
Oil field D wellhead water (DWH)e,f | 52-53g | Yes | 1 (14) | Kosmotogai | 1 (6) | M1i | 1 (1) | Kosmotogai | NA | NA | NA | NA |
Oil field D FWKO water (DF)f,h | 20-30 | Yes | 1 (45) | Kosmotogai | 1 (17) | M1i | — | — | + | + | − | M1, Kosmotoga, Petrotoga |
Oil field H FWKO water (HF)j | 30-32 | Yes | 7 (59) | M1, M2, M3, M4, Kosmotoga | 1 (29) | M1 | — | — | + | + | − | M1, Petrotoga |
Oil field H satellite water (HSAT)e,j | 41 and 50g | Yes | 1 (8) | M1 | — | — | 2 (16) | Kosmotoga, Thermotoga | NA | NA | NA | NA |
Oil field H wellhead water (HWH)e,j | 41 and 50g | Yes | NA | — | — | NA | NA | NA | + | + | + | M1, Petrotoga |
7.
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).
Open in a separate windowaConditions are at an intensity of 150 W m−2 unless otherwise indicated.bThe values reported are means ± standard errors of the means from triplicate experiments.cAdditional experiments for this condition were performed using natural freshwater.The highly resistant A. polyphaga cysts and B. subtilis spores were unaffected by SODIS or SODIS-R at an optical irradiance of 150 W m−2. However, a significant reduction in cyst viability was observed at 6 h when the optical irradiance was increased to 250 W m−2 for SODIS-R only (P < 0.001; Table Table1).1). For spores, a kill was obtained only at 320 W m−2 after 6-h exposure, and no difference between SODIS and SODIS-R was observed (Table (Table1).1). Previously, we reported a >2-log kill at 6 h for Acanthamoeba cysts by using SODIS at the higher optical irradiance of 850 W m−2, compared to the 0.1-log10 kill observed here using the lower intensity of 250 W m−2 or the 3.5-log10 kill with SODIS-R.Inactivation experiments performed with Acanthamoeba cysts and trophozoites suspended in natural freshwater gave results comparable to those obtained with Ringer''s solution (P > 0.05; Table Table1).1). However, it is acknowledged that the findings of this study are based on laboratory-grade water and freshwater and that differences in water quality through changes in turbidity, pH, and mineral composition may significantly affect the performance of SODIS (20). Accordingly, further studies are indicated to evaluate the enhanced efficacy of SODIS-R by using natural waters of varying composition in the areas where SODIS is to be employed.Previous studies with SODIS under laboratory conditions have employed lamps delivering an optical irradiance of 850 W m−2 to reflect typical natural sunlight conditions (6, 11, 12, 15, 16). Here, we used an optical irradiance of 150 to 320 W m−2 to obtain slower organism inactivation and, hence, determine the potential enhancing effect of riboflavin on SODIS.In conclusion, this study has shown that the addition of riboflavin significantly enhances the efficacy of simulated SODIS against a range of microorganisms. The precise mechanism by which photoactivated riboflavin enhances antimicrobial activity is unknown, but studies have indicated that the process may be due, in part, to the generation of singlet oxygen, H2O2, superoxide, and hydroxyl free radicals (10). Further studies are warranted to assess the potential benefits from riboflavin-enhanced SODIS in reducing the incidence of gastrointestinal infection in communities where potable water is not available. 相似文献
TABLE 1.
Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)Organism | Conditiona | Log10 reduction in viability at indicated h of exposureb | |||
---|---|---|---|---|---|
1 | 2 | 4 | 6 | ||
E. coli | SODIS | 0.0 ± 0.0 | 0.2 ± 0.1 | 5.7 ± 0.0 | 5.7 ± 0.0 |
SODIS-R | 1.1 ± 0.0 | 5.7 ± 0.0 | 5.7 ± 0.0 | 5.7 ± 0.0 | |
L. pneumophila | SODIS | 0.7 ± 0.2 | 1.3 ± 0.3 | 4.8 ± 0.2 | 4.8 ± 0.2 |
SODIS-R | 4.4 ± 0.0 | 4.4 ± 0.0 | 4.4 ± 0.0 | 4.4 ± 0.0 | |
P. aeruginosa | SODIS | 0.7 ± 0.0 | 1.8 ± 0.0 | 4.9 ± 0.0 | 4.9 ± 0.0 |
SODIS-R | 5.0 ± 0.0 | 5.0 ± 0.0 | 5.0 ± 0.0 | 5.0 ± 0.0 | |
S. aureus | SODIS | 0.0 ± 0.0 | 0.0 ± 0.0 | 6.2 ± 0.0 | 6.2 ± 0.0 |
SODIS-R | 0.2 ± 0.1 | 6.3 ± 0.0 | 6.3 ± 0.0 | 6.3 ± 0.0 | |
C. albicans | SODIS | 0.2 ± 0.0 | 0.4 ± 0.1 | 0.5 ± 0.1 | 1.0 ± 0.1 |
SODIS-R | 0.1 ± 0.0 | 0.7 ± 0.1 | 5.3 ± 0.0 | 5.3 ± 0.0 | |
F. solani conidia | SODIS | 0.2 ± 0.1 | 0.3 ± 0.0 | 0.2 ± 0.0 | 0.7 ± 0.1 |
SODIS-R | 0.3 ± 0.1 | 0.8 ± 0.1 | 1.3 ± 0.1 | 4.4 ± 0.0 | |
B. subtilis spores | SODIS | 0.3 ± 0.0 | 0.2 ± 0.0 | 0.0 ± 0.0 | 0.1 ± 0.0 |
SODIS-R | 0.1 ± 0.1 | 0.2 ± 0.1 | 0.3 ± 0.3 | 0.1 ± 0.0 | |
SODIS (250 W m−2) | 0.1 ± 0.0 | 0.1 ± 0.1 | 0.1 ± 0.1 | 0.0 ± 0.0 | |
SODIS-R (250 W m−2) | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.2 ± 0.0 | 0.4 ± 0.0 | |
SODIS (320 W m−2) | 0.1 ± 0.1 | 0.1 ± 0.0 | 0.0 ± 0.1 | 4.3 ± 0.0 | |
SODIS-R (320 W m−2) | 0.1 ± 0.0 | 0.1 ± 0.1 | 0.9 ± 0.0 | 4.3 ± 0.0 | |
A. polyphaga trophozoites | SODIS | 0.4 ± 0.2 | 0.6 ± 0.1 | 0.6 ± 0.2 | 0.4 ± 0.1 |
SODIS-R | 0.3 ± 0.1 | 1.3 ± 0.1 | 2.3 ± 0.4 | 3.1 ± 0.2 | |
SODIS, naturalc | 0.3 ± 0.1 | 0.4 ± 0.1 | 0.5 ± 0.2 | 0.3 ± 0.2 | |
SODIS-R, naturalc | 0.2 ± 0.1 | 1.0 ± 0.2 | 2.2 ± 0.3 | 2.9 ± 0.3 | |
A. polyphaga cysts | SODIS | 0.4 ± 0.1 | 0.1 ± 0.3 | 0.3 ± 0.1 | 0.4 ± 0.2 |
SODIS-R | 0.4 ± 0.2 | 0.3 ± 0.2 | 0.5 ± 0.1 | 0.8 ± 0.3 | |
SODIS (250 W m−2) | 0.0 ± 0.1 | 0.2 ± 0.3 | 0.2 ± 0.1 | 0.1 ± 0.2 | |
SODIS-R (250 W m−2) | 0.4 ± 0.2 | 0.3 ± 0.2 | 0.8 ± 0.1 | 3.5 ± 0.3 | |
SODIS (250 W m−2), naturalc | 0.0 ± 0.3 | 0.2 ± 0.1 | 0.1 ± 0.1 | 0.2 ± 0.1 | |
SODIS-R (250 W m−2), naturalc | 0.1 ± 0.1 | 0.2 ± 0.2 | 0.6 ± 0.1 | 3.4 ± 0.2 |
8.
Vertebrate genomic assemblies were analyzed for endogenous sequences related to any known viruses with single-stranded DNA genomes. Numerous high-confidence examples related to the Circoviridae and two genera in the family Parvoviridae, the parvoviruses and dependoviruses, were found and were broadly distributed among 31 of the 49 vertebrate species tested. Our analyses indicate that the ages of both virus families may exceed 40 to 50 million years. Shared features of the replication strategies of these viruses may explain the high incidence of the integrations.It has long been appreciated that retroviruses can contribute significantly to the genetic makeup of host organisms. Genes related to certain other viruses with single-stranded RNA genomes, formerly considered to be most unlikely candidates for such contribution, have recently been detected throughout the vertebrate phylogenetic tree (1, 6, 13). Here, we report that viruses with single-stranded DNA (ssDNA) genomes have also contributed to the genetic makeup of many organisms, stretching back as far as the Paleocene period and possibly the late Cretaceous period of evolution.Determining the evolutionary ages of viruses can be problematic, as their mutation rates may be high and their replication may be rapid but also sporadic. To establish a lower age limit for currently circulating ssDNA viruses, we analyzed 49 published vertebrate genomic assemblies for the presence of sequences derived from the NCBI RefSeq database of 2,382 proteins from known viruses in this category, representing a total of 23 classified genera from 7 virus families. Our survey uncovered numerous high-confidence examples of endogenous sequences related to the Circoviridae and to two genera in the family Parvoviridae: the parvoviruses and dependoviruses (Fig. (Fig.11).Open in a separate windowFIG. 1.Phylogenetic tree of vertebrate organisms and history of ssDNA virus integrations. Times of integration of ancestral dependoviruses (yellow icosahedrons), parvoviruses (blue icosahedrons), and circoviruses (triangles) are approximate.The Dependovirus and Parvovirus genomes are typically 4 to 6 kb in length, include 2 major open reading frames (encoding replicase proteins [Rep and NS1, respectively] and capsid proteins [Cap and VP1, respectively]), and have characteristic hairpin structures at both ends (Fig. (Fig.2).2). For replication, these viruses depend on host enzymes that are recruited by the viral replicase proteins to the hairpin regions, where self-primed viral DNA synthesis is initiated (2). Circovirus genomes are typically ∼2-kb circles. DNA of the type species, porcine circovirus 1 (PCV-1), contains a stem-loop structure within the origin of replication (Fig. (Fig.2),2), and the largest open reading frame includes sequences that are homologous to the Parvovirus replicase open reading frame (9, 11). The circoviruses also depend on host enzymes for replication, and DNA synthesis is self-primed from a 3′-OH end formed by endonucleolytic cleavage of the stem-loop structure (4). The frequency of Dependovirus infection is estimated to be as high as 90% within an individual''s lifetime. None of the dependoviruses have been associated with human disease, but related viruses in the family Parvoviridae (e.g., erythrovirus B19 and possibly human bocavirus) are pathogenic for humans, and members of both the Parvoviridae and the Circoviridae can cause a variety of animal diseases (2, 4).Open in a separate windowFIG. 2.Schematics illustrating the structure and organization of Parvoviridae and Circoviridae genomes and origins of several of the longest-integrated ancestral viral sequences found in vertebrates. Integrations were aligned to the Dependovirus adeno-associated virus 2 (AAV2), the Parvovirus minute virus of mice (MVM), and the Circovirus porcine circovirus 1 (PCV-1). The inverted terminal repeat (ITR) sequences in the Dependovirus and Parvovirus genomes are depicted on an expanded scale. A linear representation of the circular genome of PCV-1 is shown with the 10-bp stem-loop structure on an expanded scale. Horizontal lines beneath the maps indicate the lengths of similar sequences that could be identified by BLAST. The numbers indicate the locations of amino acids in the viral proteins where the sequence similarities in the endogenous insertions start and end. The actual ancestral virus-derived integrated sequences may extend beyond the indicated regions.With some ancestral endogenous sequences that we identified, phylogenetic comparisons can be used to estimate age. For example, as a Dependovirus-like sequence is present at the same location in the genomes of mice and rats, the ancestral virus must have existed before their divergence, more than 20 million years ago. Some Circovirus- and Dependovirus-related integrations also predate the split between dog and panda, about 42 million years ago. However, in most other cases, we rely on an indirect method for estimating age (1). As genomic sequences evolve, they accumulate new stop codons and insertion/deletion-induced frameshifts. The rates of these events can be tied directly to the rates of neutral sequence drift and, therefore, the time of evolution. To apply this method, we first performed a BLAST search of vertebrate genomes for all known ssDNA virus proteins (BLAST options, -p tblastn -M BLOSUM62 -e 1e−4). Candidate sequences were then recorded, along with 5 kb of flanking regions, and then again aligned against the database of ssDNA viruses to find the most complete alignment (BLAST options, -t blastx -F F -w 15 -t 1500 -Z 150 -G 13 -E 1 -e 1e−2). Detected alignments were then compared with a neutral model of genome evolution, as described in the supplemental material, and the numbers of stop codons and frameshifts were converted into the expected genomic drift undergone by the sequences. The age of integration was then estimated from the known phylogeny of vertebrates (7, 10). Using these methods, we discovered that as many as 110 ssDNA virus-related sequences have been integrated into the 49 vertebrate genomes considered, during a time period ranging from the present to over 40 to 60 million years ago (Table (Table1;1; see also Tables S1 to S3 in the supplemental material).
Open in a separate windowaSome ambiguity in choosing the most similar virus is possible. We generally used the alignment with the lowest E value in the BLAST results. However, one or two points in the exponent of an E value were sometimes sacrificed to achieve a longer sequence alignment.baa, amino acids.cThese sequences have long insertions compared to the present-day viruses. In all cases tested, these insertions originated from short interspersed elements (SINEs). These insertions were excluded from the counts of stop codons and frameshifts and the estimation of integration age.dChr, chromosome.It is important to recognize that there is an intrinsic limit on how far back in time we can reach to identify ancient endogenous viral sequences. First, the sequences must be identified with confidence by BLAST or similar programs. This requirement places a lower limit on sequence identity at about 20 to 30% of amino acids, or about 75% of nucleotides (nucleotides evolve nearly 2.5 times slower than the amino acid sequence they encode). Second, the related, present-day virus must have evolved at a rate that is not much higher than that of the endogenous sequences. The viruses for which ancestral endogenous sequences were identified in this study exhibit sequence drift similar to that associated with mammalian genomes. Setting this rate at 0.14% per million years of evolution (8), we arrive at 90 million years as the theoretical limit for the oldest sequences that can be identified using our methods. This limit drops to less than 35 million years for endogenous viral sequences in rodents and even lower for sequences related to viruses that evolve faster than mammalian genomes.The most widespread integrations found in our survey are derived from the dependoviruses. These include nearly complete genomes related to adeno-associated virus (AAV) in microbat, wallaby, dolphin, rabbit, mouse, and baboon (Fig. (Fig.2).2). We did not detect inverted terminal repeats in several integrations tested, even though repeats are common in the present-day dependoviruses. This result could be explained by sequence decay or the absence of such structures in the ancestral viruses. However, we do see sequences that resemble degraded hairpin structures to which Dependovirus Rep proteins bind, with an example from microbat integration mlEDLG-1 shown in Fig. Fig.3.3. The second most widespread endogenous sequences are related to the parvoviruses. They are found in 6 of 49 vertebrate species considered, with nearly complete genomes in rat, opossum, wallaby, and guinea pig (Fig. (Fig.22).Open in a separate windowFIG. 3.Hairpin structure of the inverted terminal repeat of adeno-associated virus 2 (left) and a candidate degraded hairpin structure located close to the 5′ end of the mlEDLG-1 integration in microbats (right). Structures and mountain plots were generated using default parameters of the RNAfold program (5), with nucleotide coloring representing base-pairing probabilities: blue is below average, green is average, and red is above average. Mountain plots represent hairpin structures based on minimum free energy (mfe) calculations and partition function (pf) calculations, as well as the centroid structure (5). Height is expressed in numbers of nucleotides; position represents nucleotide.The Dependovirus AAV2 has strong bias for integration into human chromosome 19 during infection, driven by a host sequence that is recognized by the viral Rep protein(s). Rep mediates the formation of a synapse between viral and cellular sequences, and the cellular sequences are nicked to serve as an origin of viral replication (14). The related integrations in mice and rats, located in the same chromosomal locations, might be explained by such a mechanism. However, the extent of endogenous sequence decay and the frequency of stop codons indicate that these integrations occurred some 30 to 35 million years ago, implying that they are derived from a single event in a rodent ancestor rather than two independent integration events at the same location. Similarly, integrations EDLG-1 in dog and panda lie in chromosomal regions that can be readily aligned (based on University of California—Santa Cruz [UCSC] genome assemblies) and show sequence decay consistent with the age of the common ancestor, about 42 million years. Endogenous sequences related to the family Parvoviridae can thus be traced to over 40 million years back in time, and viral proteins related to this family have remained over 40% conserved.Sequences related to circoviruses were detected in five vertebrate species (Table (Table11 and Table S1 in the supplemental material). At least one of these sequences, the endogenous sequence in opossum, likely represents a recent integration. Several integrations in dog, cat, and panda, on the other hand, appear to date from at least 42 million years ago, which is the last time when pandas and dogs shared a common ancestor. We see evidence for this age in data from sequence degradation (Table (Table1),1), phylogenetic analyses of endogenous Circovirus-like genomes (see Fig. S2 in the supplemental material), and genomic synteny where integration ECLG-3 is surrounded by genes MTA3 and ARID5A in both dog and panda and integration ECLG-2 lies 35 to 43 kb downstream of gene UPF3A. In fact, Circovirus integrations may even precede the split between dogs and cats, about 55 million years ago, although the preliminary assembly and short genomic contigs for cats make synteny analysis impossible.The most common Circovirus-related sequences detected in vertebrate genomes are derived from the rep gene. We speculate that, like those of the Parvoviridae, the ancestral Circoviridae sequences might have been copied using a primer sequence in the host DNA that resembled the viral origin and was therefore recognized by the virus Rep protein. Higher incidence of rep gene identifications may represent higher conservation of this gene with time, or alternatively, possession of these sequences may impart some selective advantage to the host species. The largest Circovirus-related integration detected, in the opossum, comprises a short fragment of what may have been the cap gene immediately adjacent to and in the opposite orientation from the rep gene. This organization is similar to that of the present day Circovirus genome in which these genes share a promoter in the hairpin regions but are translated in opposite directions (Fig. (Fig.22).In summary, our results indicate that sequences derived from ancestral members of the families Parvoviridae and Circoviridae were integrated into their host''s genomes over the past 50 million years of evolution. Features of their replication strategies suggest mechanisms by which such integrations may have occurred. It is possible that some of the endogenous viral sequences could offer a selective advantage to the virus or the host. We note that rep open reading frame-derived proteins from some members of these families kill tumor cells selectively (3, 12). The genomic “fossils” we have discovered provide a unique glimpse into virus evolution but can give us only a lower estimate of the actual ages of these families. However, numerous recent integrations suggest that their germ line transfer has been continuing into present times. 相似文献
TABLE 1.
Selected endogenous sequences in vertebrate genomes related to single-stranded DNA virusesVirus group and vertebrate species | Initial genomic search using TBLASTN | Best sequence homology identified using BLASTX | Predicted nucleotide drift (%) | Integration label | Age (million yr) or timing of integration based on sequence aging | |||||
---|---|---|---|---|---|---|---|---|---|---|
Chromosomal or scaffold location | Protein | BLAST E value/% sequence identity | Most similar virusa | Protein | Coordinates | No. of stop codons/frameshifts | ||||
Circoviruses | ||||||||||
Cat | Scaffold_62068 | Rep | 6E−05/37 | Canary circovirus | Rep | 4-283 | 3/7 in 268 aab | 14.2 | fcECLG-1 | 82 |
Scaffold_24038 | Rep | 6E−06/51 | Columbid circovirus | Rep | 44-317 | 4/5 in 231 aac | 15.2 | fcECLG-2 | 87 | |
Dog | Chr5d | Rep | 7E−16/46 | Raven circovirus | Rep | 16-263 | 6/5 in 250 aa | 17.6 | cfECLG-1 | 98 |
Chr22 | Rep | 1E−14/43 | Beak and feather disease virus | Rep | 7-264 | 2/1 in 261 aac | 4.5 | cfECLG-2 | 54 | |
Opossum | Chr3 | Rep | 4E−46/44 | Finch circovirus | Rep | 2-291 | 0/2 in 282 aa | 2.3 | mdECLG | 12 |
Cap | 6-36 | 0/0 in 30 aa | ||||||||
Dependoviruses | ||||||||||
Dog | ChrX | Rep | 6E−05/55 | AAV5 | Rep | 239-445 | 3/4 in 200 aa | 14.0 | cfEDLG-1 | 78 |
Dolphin | GeneScaffold1475 | Rep | 8E−39/39 | Avian AAV DA1 | Rep | 79-486 | 3/4 in 379 aac | 6.6 | ttEDLG-2 | 55 |
Cap | 4E−61/47 | Cap | 1-738 | 4/7 in 678 aac | ||||||
Elephant | Scaffold_4 | Rep | 0/55 | AAV5 | Rep | 3-589 | 0/0 in 579 aa | 0.0 | laEDLG | Recent |
Hyrax | GeneScaffold5020 | Cap | 3E−34/53 | AAV3 | Cap | 485-735 | 0/5 in 256 aa | 7.0 | pcEDLG-1 | 29 |
Scaffold_19252 | Rep | 9E−72/47 | Bovine AAV | Rep | 2-348 | 8/4 in 348 aa | 14.3 | pcEDLG-2 | 60 | |
Megabat | Scaffold_5601 | Rep | 2E−13/31 | AAV2 | Rep | 315-479 | 1/5 in 175 aa | 13.1 | pvEDLG-3 | 76 |
Microbat | GeneScaffold2026 | Rep | 1E−117/50 | AAV2 | Rep | 1-617 | 2/5 in 612 aa | 5.8 | mlEDLG-1 | 27 |
Cap | 9E−33/51 | Cap | 1-731 | 2/9 in 509 aac | ||||||
Scaffold_146492 | Cap | 6E−32/42 | AAV2 | Cap | 479-732 | 0/3 in 252 aa | 4.2 | mlEDLG-2 | 19 | |
Mouse | Chr1 | Rep | 2E−06/34 | AAV2 | Rep | 4-206 | 3/5 in 191 aa | 17.1 | mmEDLG-1 | 39 |
Chr3 | Rep | 2E−24/31 | AAV5 | Rep | 71-478 | 12/7 in 389 aa | 16.5 | mmEDLG-2 | 37 | |
Cap | 2E−22/45 | Cap | 22-724 | 12/10 in 649aac | ||||||
Chr8 | Rep | 1E−08/46 | AAV2 | Rep | 314-473 | 3/3 in 147 aa | 13.8 | mmEDLG-3 | 31 | |
Cap | 1-137 | 1/2 in 114 aa | ||||||||
Panda | Scaffold2359 | Rep | 2E−06/37 | Bovine AAV | Rep | 238-426 | 2/3 in 186 aa | 10.4 | amEDLG-1 | 59 |
Pika | Scaffold_9941 | Rep | 4E−14/28 | AAV5 | Rep | 126-415 | 2/2 in 282 aa | 5.4 | opEDLG | 14 |
Platypus | Chr2 | Rep | 9E−10/35 | Bovine AAV | Rep | 297-437 | 4/3 in 138 aa | 17.1 | oaEDLG-1 | 79 |
Cap | 272-419 | 1/2 in 150 aac | ||||||||
Contig12430 | Rep | 2E−09/47 | Bovine AAV | Rep | 353-450 | 3/1 in 123 aa | 12.0 | oaEDLG-2 | 55 | |
Cap | 2E−05/32 | Cap | 253-367 | 2/1 in 116 aa | ||||||
Rabbit | Chr10 | Rep | 3E−97/39 | AAV2 | Rep | 1-619 | 3/9 in 613 aa | 9.3 | ocEDLG | 43 |
Cap | 5E−50/45 | Cap | 1-723 | 10/9 in 675 aa | ||||||
Rat | Chr13 | Rep | 2E−09/33 | AAV2 | Rep | 4-175 | 2/4 in 177 aa | 13.3 | rnEDLG-1 | 28 |
Chr2 | Rep | 4E−18/40 | AAV5 | Rep | 1-461 | 12/12 in 454 aa | 22.7 | rnEDLG-2 | 51 | |
Chr19 | Rep | 2E−07/33 | AAV5 | Rep | 329-464 | 2/4 in 136 aa | 16.1 | rnEDLG-3 | 35 | |
Cap | 31-133 | 2/1 in 93 aa | ||||||||
Tarsier | Scaffold_178326 | Rep | 4E−14/23 | AAV5 | Rep | 96-465 | 2/3 in 356 aa | 5.3 | tsEDLG | 23 |
Parvoviruses | ||||||||||
Guinea pig | Scaffold_188 | Rep | 3E−24/46 | Porcine parvovirus | Rep | 313-567 | 5/3 in 250 aa | 12.3 | cpEPLG-1 | 40 |
Cap | 1E−16/36 | Cap | 10-689 | 11/12 in 672 aa | ||||||
Scaffold_27 | Rep | 1E−50/39 | Canine parvovirus | Rep | 11-640 | 1/4 in 616 aa | 5.3 | cpEPLG-2 | 17 | |
Cap | 1E−38/39 | Porcine parvovirus | Cap | 3-719 | 2/14 in 700 aa | |||||
Tenrec | Scaffold_260946 | Rep | 2E−20/38 | LuIII virus | Rep | 406-598 | 4/4 in 190 aa | 19.0 | etEPLG-2 | 60 |
Cap | 11-639 | 16/15 in 595 aa | ||||||||
Rat | Chr5 | Rep | 6E−10/56 | Canine parvovirus | Rep | 1-282 | 0/0 in 312 aa | 0.6 | rnEPLG | Recent |
Cap | 0/62 | Cap | 637-667 | 0/2 in 760 aa | ||||||
Rep | 0/63 | 1-751 | ||||||||
Opossum | Chr3 | Rep | 2E−39/33 | LuIII virus | Rep | 7-570 | 11/3 in 502 aa | 10.9 | mdEPLG-2 | 56 |
Cap | 7E−8/33 | Cap | 11-729 | 14/7 in 704 aa | ||||||
Chr6 | Rep | 6E−58/44 | Porcine parvovirus | Rep | 16-563 | 3/7 in 534 aac | 4.6 | mdEPLG-3 | 24 | |
Cap | 6E−60/38 | Cap | 10-715 | 2/5 in 707 aac | ||||||
Wallaby | Scaffold_108040 | Rep | 4E−74/62 | Canine parvovirus | Rep | 341-645 | 0/0 in 287 aa | 1.3 | meEPLG-3 | 7 |
Cap | 8E−37/32 | Cap | 35-738 | 0/4 in 687 aa | ||||||
Scaffold_72496 | Rep | 2E−61/42 | Porcine parvovirus | Rep | 23-567 | 4/3 in 531 aa | 5.7 | meEPLG-6 | 30 | |
Cap | 2E−31/38 | Cap | 10-532 | 6/4 in 514 aa | ||||||
Scaffold_88340 | Rep | 7E−37/55 | Mouse parvovirus 1 | Rep | 344-566 | 0/3 in 223 aa | 6.7 | meEPLG-16 | 36 | |
Cap | 7E−22/33 | Cap | 11-713 | 6/9 in 700 aa |
9.
Banumathi Sankaran Shilah A. Bonnett Kinjal Shah Scott Gabriel Robert Reddy Paul Schimmel Dmitry A. Rodionov Valérie de Crécy-Lagard John D. Helmann Dirk Iwata-Reuyl Manal A. Swairjo 《Journal of bacteriology》2009,191(22):6936-6949
GTP cyclohydrolase I (GCYH-I) is an essential Zn2+-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ∼25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn2+-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn2+ starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn2+-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.The Zn2+-dependent enzyme GTP cyclohydrolase I (GCYH-I; EC 3.5.4.16) is the first enzyme of the de novo tetrahydrofolate (THF) biosynthesis pathway (Fig. (Fig.1)1) (38). THF is an essential cofactor in one-carbon transfer reactions in the synthesis of purines, thymidylate, pantothenate, glycine, serine, and methionine in all kingdoms of life (38), and formylmethionyl-tRNA in bacteria (7). Recently, it has also been shown that GCYH-I is required for the biosynthesis of the 7-deazaguanosine-modified tRNA nucleosides queuosine and archaeosine produced in Bacteria and Archaea (44), respectively, as well as the 7-deazaadenosine metabolites produced in some Streptomyces species (33). GCYH-I is encoded in Escherichia coli by the folE gene (28) and catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (55), a complex reaction that begins with hydrolytic opening of the purine ring at C-8 of GTP to generate an N-formyl intermediate, followed by deformylation and subsequent rearrangement and cyclization of the ribosyl moiety to generate the pterin ring in THF (Fig. (Fig.1).1). Notably, the enzyme is dependent on an essential active-site Zn2+ that serves to activate a water molecule for nucleophilic attack at C-8 in the first step of the reaction (2).Open in a separate windowFIG. 1.Reaction catalyzed by GCYH-I, and metabolic fate of 7,8-dihydroneopterin triphosphate.A homologous GCYH-I is found in mammals and other higher eukaryotes, where it catalyzes the first step of the biopterin (BH4) pathway (Fig. (Fig.1),1), an essential cofactor in the biosynthesis of tyrosine and neurotransmitters, such as serotonin and l-3,4-dihydroxyphenylalanine (3, 52). Recently, a distinct class of GCYH-I enzymes, GCYH-IB (encoded by the folE2 gene), was discovered in microbes (26% of sequenced Bacteria and most Archaea) (12), including several clinically important human pathogens, e.g., Neisseria and Staphylococcus species. Notably, GCYH-IB is absent in eukaryotes.The distribution of folE (gene product renamed GCYH-IA) and folE2 (GCYH-IB) in bacteria is diverse (12). The majority of organisms possess either a folE (65%; e.g., Escherichia coli) or a folE2 (14%; e.g., Neisseria gonorrhoeae) gene. A significant number (12%; e.g., B. subtilis) possess both genes (a subset of 50 bacterial species is shown in Table Table1),1), and 9% lack both genes, although members of the latter group are mainly intracellular or symbiotic bacteria that rely on external sources of folate. The majority of Archaea possess only a folE2 gene, and the encoded GCYH-IB appears to be necessary only for the biosynthesis of the modified tRNA nucleoside archaeosine (44) except in the few halophilic Archaea that are known to synthesize folates, such as Haloferax volcanii, where GCYH-IB is involved in both archaeosine and folate formation (13, 44).
TABLE 1.
Distribution and candidate Zur-dependent regulation of alternative GCYH-I genes in bacteriaaOpen in a separate windowaGenes that are preceded by candidate Zur binding sites.bZur-regulated cluster is on the virulence plasmid pLVPK.cExamples of organisms with no folE genes are in boldface type.dZn-dependent regulation of B. subtilis folE2 by Zur was experimentally verified (17).Expression of the Bacillus subtilis folE2 gene, yciA, is controlled by the Zn2+-dependent Zur repressor and is upregulated under Zn2+-limiting conditions (17). This led us to propose that the GCYH-IB family utilizes a metal other than Zn2+ to allow growth in Zn2+-limiting environments, a hypothesis strengthened by the observation that an archaeal ortholog from Methanocaldococcus jannaschii has recently been shown to be Fe2+ dependent (22). To test this hypothesis, we investigated the physiological role of GCYH-IB in B. subtilis, an organism that contains both isozymes, as well as the metal dependence of B. subtilis GCYH-IB in vitro. To gain a structural understanding of the metal dependence of GCYH-IB, we determined high-resolution crystal structures of Zn2+- and Mn2+-bound forms of the N. gonorrhoeae ortholog. Notably, although the GCYH-IA and -IB enzymes belong to the tunneling-fold (T-fold) superfamily, there are significant differences in their global and active-site architecture. These studies shed light on the physiological significance of the alternative folate biosynthesis isozymes in bacteria exposed to various metal environments, and offer a structural understanding of the differential metal dependence of GCYH-IA and -IB. 相似文献10.
Lisa Jacobsen Lisa Durso Tyrell Conway Kenneth W. Nickerson 《Applied and environmental microbiology》2009,75(13):4633-4635
Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table (Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table (Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table (Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table Table22.
Open in a separate window
Open in a separate windowaEight LB controls were run, two for each set of LB experiments: SDS, CTAB, benzalkonium chloride (BAC), and pH stress.bGrowth was measured as either +++, +, or 0 (good, poor, and none, respectively), with +++ being the growth achieved on the LB control plates. “Variable” means that two or three replicates did not agree. All experiments were done at 37°C.c“Anaerobic” refers to use of an Oxoid anaerobic chamber. Aerobic and anaerobic growth data are presented together when the results were identical and separately when the results were not the same or the anaerobic set had not been done. LB plates were measured after 1 (aerobic) or 2 (anaerobic) days, and the M63 plates were measured after 2 or 3 days.dCTAB used at 0.05, 0.2%, and 0.4%.eM63 defined medium (3) was supplemented with glucose, gluconate, or glucuronate, all at 0.2%.fIdentical results were obtained with and without 0.0001% thiamine.gND, not determined. 相似文献
TABLE 1.
E. coli strains used in this studyE. coli strain (n) | Source |
---|---|
ECOR strains (72) | Thomas Whittman |
Laboratory adapted (6) | |
K-12 Davis | Paul Blum |
CG5C 4401 | Paul Blum |
K-12 Stanford | Paul Blum |
W3110 | Paul Blum |
B | Tyler Kokjohn |
AB 1157 | Tyler Kokjohn |
O157:H7 (10) | |
FRIK 528 | Andrew Benson |
ATCC 43895 | Andrew Benson |
MC 1061 | Andrew Benson |
C536 | Tim Cebula |
C503 | Tim Cebula |
C535 | Tim Cebula |
ATCC 43889 | William Cray, Jr. |
ATCC 43890 | William Cray, Jr. |
ATCC 43888 | Willaim Cray, Jr. |
ATCC 43894 | William Cray, Jr. |
TABLE 2.
Physiological comparison of 88 strains of Escherichia coliGrowth medium or condition | Oxygenc | No. of strains with type of growthb
| |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ECOR strains (n = 72)
| Laboratory strains (n = 6)
| O157:H7 strains (n = 10)
| |||||||||||
Good | Poor | None | Variable | Good | Poor | None | Variable | Good | Poor | None | Variable | ||
LB controla | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
1% SDS | Aerobic | 69 | 3 | 0 | 0 | 6 | 0 | 0 | 0 | 8 | 0 | 0 | 2 |
5% SDS | Aerobic | 68 | 4 | 0 | 0 | 6 | 0 | 0 | 0 | 8 | 2 | 0 | 0 |
1% SDS | Anaerobic | 53 | 15 | 4 | 0 | 2 | 3 | 1 | 0 | 1 | 7 | 0 | 2 |
5% SDS | Anaerobic | 0 | 68 | 4 | 0 | 0 | 4 | 2 | 0 | 0 | 7 | 0 | 4 |
CTABd (all) | Both | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 |
0.05% BAC | Aerobic | 3 | 11 | 58 | 2 | 0 | 2 | 2 | 2 | 0 | 0 | 9 | 1 |
0.2% BAC | Aerobic | 0 | 1 | 71 | 0 | 1 | 0 | 5 | 0 | 0 | 0 | 10 | 0 |
0.05% BAC | Anaerobic | 2 | 3 | 67 | 0 | 0 | 1 | 5 | 0 | 0 | 0 | 9 | 1 |
0.2% BAC | Anaerobic | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 |
pH 6.5 | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 6 | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 5 | Both | 70 | 2 | 0 | 0 | 6 | 0 | 0 | 0 | 9 | 0 | 0 | 1 |
pH 4.6 | Both | 70 | 2 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 4.3 | Aerobic | 14 | 0 | 1 | 57 | 3 | 1 | 2 | 0 | 3 | 2 | 0 | 5 |
pH 4.3 | Anaerobic | 69 | 3 | 0 | 0 | 3 | 1 | 2 | 0 | 1 | 1 | 0 | 0 |
pH 4.1 or 4.2 | Aerobic | 0 | 0 | 72 | 0 | NDg | ND | ||||||
pH 4.0 | Both | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 9 | 1 |
M63 with supplemente | |||||||||||||
Glucose | Aerobicf | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucose | Anaerobicf | 70 | 0 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Gluconate | Both | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucuronate | Aerobic | 68 | 2 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucuronate | Anaerobic | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
11.
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13.
Canada geese (Branta canadensis) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-Bac and CGOF2-Bac) allowing for the specific and sensitive detection of Bacteroides 16S rRNA gene markers present within Canada goose feces.The Canada goose (Branta canadensis) is a prevalent waterfowl species in North America. The population density of Canada geese has doubled during the past 15 years, and the population was estimated to be close to 3 million in 2007 (4). Canada geese often congregate within urban settings, likely due to available water sources, predator-free grasslands, and readily available food supplied by humans (6). They are suspected to contribute to pollution of aquatic environments due to the large amounts of fecal matter that can be transported into the water. This can create a public health threat if the fecal droppings contain pathogenic microorganisms (6, 7, 9, 10, 12, 13, 19). Therefore, tracking transient fecal pollution of water due to fecal inputs from waterfowl, such as Canada geese, is of importance for protecting public health.PCR detection of host-specific 16S rRNA gene sequences from Bacteroidales of fecal origin has been described as a promising microbial source-tracking (MST) approach due to its rapidity and high specificity (2, 3). Recently, Lu et al. (15) characterized the fecal microbial community from Canada geese by constructing a 16S rRNA gene sequence database using primers designed to amplify all bacterial 16S rRNA gene sequences. The authors reported that the majority of the 16S rRNA gene sequences obtained were related to Clostridia or Bacilli and to a lesser degree Bacteroidetes, which represent possible targets for host-specific source-tracking assays.The main objective of this study was to identify novel Bacteroidales 16S rRNA gene sequences that are specific to Canada goose feces and design primers and TaqMan fluorescent probes for sensitive and specific quantification of Canada goose fecal contamination in water sources.Primers 32F and 708R from Bernhard and Field (2) were used to construct a Bacteroidales-specific 16S rRNA gene clone library from Canada goose fecal samples (n = 15) collected from grass lawns surrounding Wascana Lake (Regina, SK, Canada) in May 2009 (for a detailed protocol, see File S1 in the supplemental material). Two hundred eighty-eight clones were randomly selected and subjected to DNA sequencing (at the Plant Biotechnology Institute DNA Technologies Unit, Saskatoon, SK, Canada). Representative sequences of each operational taxonomic unit (OTU) were recovered using an approach similar to that described by Mieszkin et al. (16). Sequences that were less than 93% similar to 16S rRNA gene sequences from nontarget host species in GenBank were used in multiple alignments to identify regions of DNA sequence that were putatively goose specific. Subsequently, two TaqMan fluorescent probe sets (targeting markers designated CGOF1-Bac and CGOF2-Bac) were designed using the RealTimeDesign software provided by Biosearch Technologies (http://www.biosearchtech.com/). The newly designed primer and probe set for the CGOF1-Bac assay included CG1F (5′-GTAGGCCGTGTTTTAAGTCAGC-3′) and CG1R (5′-AGTTCCGCCTGCCTTGTCTA-3′) and a TaqMan probe (5′-6-carboxyfluorescein [FAM]-CCGTGCCGTTATACTGAGACACTTGAG-Black Hole Quencher 1 [BHQ-1]-3′), and the CGOF2-Bac assay had primers CG2F (5′-ACTCAGGGATAGCCTTTCGA-3′) and CG2R (5′-ACCGATGAATCTTTCTTTGTCTCC-3′) and a TaqMan probe (5′-FAM-AATACCTGATGCCTTTGTTTCCCTGCA-BHQ-1-3′). Oligonucleotide specificities for the Canada goose-associated Bacteroides 16S rRNA primers were verified through in silico analysis using BLASTN (1) and the probe match program of the Ribosomal Database Project (release 10) (5). Host specificity was further confirmed using DNA extracts from 6 raw human sewage samples from various geographical locations in Saskatchewan and 386 fecal samples originating from 17 different animal species in Saskatchewan, including samples from Canada geese (n = 101) (Table (Table1).1). An existing nested PCR assay for detecting Canada goose feces (15) (targeting genetic marker CG-Prev f5) (see Table S1 in the supplemental material) was also tested for specificity using the individual fecal and raw sewage samples (Table (Table1).1). All fecal DNA extracts were obtained from 0.25 g of fecal material by using the PowerSoil DNA extraction kit (Mo Bio Inc., Carlsbad, CA) (File S1 in the supplemental material provides details on the sample collection).
Open in a separate windowaThe 6 goose samples that tested negative for the All-Bac marker also tested negative for the three goose markers.The majority of the Canada goose feces analyzed in this study (94%; 95 of 101) carried the Bacteroidales order-specific genetic marker designated All-Bac, with a relatively high median concentration of 8.2 log10 copies g−1 wet feces (Table (Table11 and Fig. Fig.1).1). The high prevalence and abundance of Bacteroidales in Canada goose feces suggested that detecting members of this order could be useful in identifying fecal contamination associated with Canada goose populations.Open in a separate windowFIG. 1.Concentrations of the Bacteroidales (All-Bac, CGOF1-Bac, and CGOF2-Bac) genetic markers in feces from various individual Canada geese.The composition of the Bacteroidales community in Canada goose feces (n = 15) was found to be relatively diverse since 52 OTUs (with a cutoff of 98% similarity) were identified among 211 nonchimeric 16S rRNA gene sequences. Phylogenetic analysis of the 52 OTUs (labeled CGOF1 to CGOF52) revealed that 43 (representing 84% of the 16S rRNA gene sequences) were Bacteroides like and that 9 (representing 16% of the 16S rRNA gene sequences) were likely to be members of the Prevotella-specific cluster (see Fig. S2 in the supplemental material). Similarly, Jeter et al. (11) reported that 75.7% of the Bacteroidales 16S rRNA clone library sequences generated from goose fecal samples were Bacteroides like. The majority of the Bacteroides- and Prevotella-like OTUs were dispersed among a wide range of previously characterized sequences from various hosts and did not occur in distinct clusters suitable for the design of Canada goose-associated real-time quantitative PCR (qPCR) assays (see Fig. S2 in the supplemental material). However, two single Bacteroides-like OTU sequences (CGOF1 and CGOF2) contained putative goose-specific DNA regions that were identified by in silico analysis (using BLASTN, the probe match program of the Ribosomal Database Project, and multiple alignment). The primers and probe for the CGOF1-Bac and CGOF2-Bac assays were designed with no mismatches to the clones CGOF1 and CGOF2, respectively.The CGOF2-Bac assay demonstrated no cross-amplification with fecal DNA from other host groups, while cross-amplification for the CGOF1-Bac assay was limited to one pigeon fecal sample (1 of 25, i.e., 4% of the samples) (Table (Table1).1). Since the abundance in the pigeon sample was low (3.3 log10 marker copies g−1 feces) and detection occurred late in the qPCR (with a threshold cycle [CT] value of 37.1), it is unlikely that this false amplification would negatively impact the use of the assay as a tool for detection of Canada goose-specific fecal pollution in environmental samples. In comparison, the nested PCR CG-Prev f5 assay described by Lu and colleagues (15) demonstrated non-host-specific DNA amplification with fecal DNA samples from several animals, including samples from humans, pigeons, gulls, and agriculturally relevant pigs and chickens (Table (Table11).Both CGOF1-Bac and CGOF2-Bac assays showed limits of quantification (less than 10 copies of target DNA per reaction) similar to those of other host-specific Bacteroidales real-time qPCR assays (14, 16, 18). The sensitivities of the CGOF1-Bac and CGOF2-Bac assays were 57% (with 58 of 101 samples testing positive) and 50% (with 51 of 101 samples testing positive) for Canada goose feces, respectively (Table (Table1).1). A similar sensitivity of 58% (with 59 of 101 samples testing positive) was obtained using the CG-Prev f5 PCR assay. The combined use of the three assays increased the detection level to 72% (73 of 101) (Fig. (Fig.2).2). Importantly, all markers were detected within groups of Canada goose feces collected each month from May to September, indicating relative temporal stability of the markers. The CG-Prev f5 PCR assay is an end point assay, and therefore the abundance of the gene marker in Canada goose fecal samples could not be determined. However, development of the CGOF1-Bac and CGOF2-Bac qPCR approach allowed for the quantification of the host-specific CGOF1-Bac and CGOF2-Bac markers. In the feces of some individual Canada geese, the concentrations of CGOF1-Bac and CGOF2-Bac were high, reaching levels up to 8.8 and 7.9 log10 copies g−1, respectively (Fig. (Fig.11).Open in a separate windowFIG. 2.Venn diagram for Canada goose fecal samples testing positive with the CGOF1-Bac, CGOF2-Bac, and/or CG-Prev f5 PCR assay. The number outside the circles indicates the number of Canada goose fecal samples for which none of the markers were detected.The potential of the Canada goose-specific Bacteroides qPCR assays to detect Canada goose fecal pollution in an environmental context was tested using water samples collected weekly during September to November 2009 from 8 shoreline sampling sites at Wascana Lake (see File S1 and Fig. S1 in the supplemental material). Wascana Lake is an urban lake, located in the center of Regina, that is routinely frequented by Canada geese. In brief, a single water sample of approximately 1 liter was taken from the surface water at each sampling site. Each water sample was analyzed for Escherichia coli enumeration using the Colilert-18/Quanti-Tray detection system (IDEXX Laboratories, Westbrook, ME) (8) and subjected to DNA extraction (with a PowerSoil DNA extraction kit [Mo Bio Inc., Carlsbad, CA]) for the detection of Bacteroidales 16S rRNA genetic markers using the Bacteroidales order-specific (All-Bac) qPCR assay (14), the two Canada goose-specific (CGOF1-Bac and CGOF2-Bac) qPCR assays developed in this study, and the human-specific (BacH) qPCR assay (17). All real-time and conventional PCR procedures as well as subsequent data analysis are described in the supplemental material and methods. The E. coli and All-Bac quantification data demonstrated that Wascana Lake was regularly subjected to some form of fecal pollution (Table (Table2).2). The All-Bac genetic marker was consistently detected in high concentrations (6 to 7 log10 copies 100 ml−1) in all the water samples, while E. coli concentrations fluctuated according to the sampling dates and sites, ranging from 0 to a most probable number (MPN) of more than 2,000 100 ml−1. High concentrations of E. coli were consistently observed when near-shore water experienced strong wave action under windy conditions or when dense communities of birds were present at a given site and time point.
Open in a separate windowaMin, minimum; max, maximum.The frequent detection of the genetic markers CGOF1-Bac (in 65 of 75 water samples [87%]), CGOF2-Bac (in 55 of 75 samples [73%]), and CG-Prev f5 (in 60 of 75 samples [79%]) and the infrequent detection of the human-specific Bacteroidales 16S rRNA gene marker BacH (17) (in 5 of 75 water samples [7%[) confirmed that Canada geese significantly contributed to the fecal pollution in Wascana Lake during the sampling period. Highest mean concentrations of both CGOF1-Bac and CGOF2-Bac markers were obtained at the sampling sites W3 (3.8 and 3.9 log10 copies 100 ml−1) and W4 (3.4 log10 copies 100 ml−1 for both), which are heavily frequented by Canada geese (Table (Table2),2), further confirming their significant contribution to fecal pollution at these particular sites. It is worth noting that concentrations of the CGOF1-Bac and CGOF2-Bac markers in water samples displayed a significant positive relationship with each other (correlation coefficient = 0.87; P < 0.0001), supporting the accuracy of both assays for identifying Canada goose-associated fecal pollution in freshwater.In conclusion, the CGOF1-Bac and CGOF2-Bac qPCR assays developed in this study are efficient tools for estimating freshwater fecal inputs from Canada goose populations. Preliminary results obtained during the course of the present study also confirmed that Canada geese can serve as reservoirs of Salmonella and Campylobacter species (see Fig. S3 in the supplemental material). Therefore, future work will investigate the cooccurence of these enteric pathogens with the Canada goose fecal markers in the environment. 相似文献
TABLE 1.
Specificities of the CGOF1-Bac, CGOF2-Bac, and CG-Prev f5 PCR assays for different species present in Saskatchewan, CanadaHost group or sample type | No. of samples | No. positive for Bacteroidales marker: | |||
---|---|---|---|---|---|
CGOF1-Bac | CGOF2-Bac | CG-Prev f5 | All-Bac | ||
Individual human feces | 25 | 0 | 0 | 1 | 25 |
Raw human sewage | 6 | 0 | 0 | 0 | 6 |
Cows | 41 | 0 | 0 | 0 | 41 |
Pigs | 48 | 0 | 0 | 1 | 48 |
Chickens | 34 | 0 | 0 | 8 | 34 |
Geese | 101 | 58 | 51 | 59 | 95a |
Gulls | 16 | 0 | 0 | 6 | 14 |
Pigeons | 25 | 1 | 0 | 2 | 22 |
Ducks | 10 | 0 | 0 | 0 | 10 |
Swans | 1 | 0 | 0 | 0 | 1 |
Moose | 10 | 0 | 0 | 0 | 10 |
Deer | |||||
White tailed | 10 | 0 | 0 | 0 | 10 |
Mule | 10 | 0 | 0 | 0 | 10 |
Fallow | 10 | 0 | 0 | 0 | 10 |
Caribou | 10 | 0 | 0 | 0 | 10 |
Bison | 10 | 0 | 0 | 0 | 10 |
Goats | 10 | 0 | 0 | 0 | 10 |
Horses | 15 | 0 | 0 | 0 | 15 |
Total | 392 | 59 | 51 | 77 | 381 |
TABLE 2.
Levels of E. coli and incidences of the Canada goose-specific (CGOF1-Bac and CGOF2-Bac), human-specific (BacH), and generic (All-Bac) Bacteroidales 16S rRNA markers at the different Wascana Lake sites sampled weeklyaSite | E. coli | All-Bac | CGOF1-Bac | CGOF2-Bac | BacH | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
No. of positive water samples/total no. of samples analyzed (%) | Min level-max level (MPN 100 ml−1) | Mean level (MPN 100 ml−1) | No. of positive water samples/total no. of samples analyzed (%) | Min level-max level (log copies 100 ml−1) | Mean level (log copies 100 ml−1) | No. of positive water samples/total no. of samples analyzed (%) | Min level-max level (log copies 100 ml−1) | Mean level (log copies 100 ml−1) | No. of positive water samples/total no. of samples analyzed (%) | Min level-max level (log copies 100 ml−1) | Mean level (log copies 100 ml−1) | No. of positive water samples/total no. of samples analyzed | Min level-max level (log copies 100 ml−1) | Mean level (log copies 100 ml−1) | |
W1 | 8/8 (100) | 6-196 | 71.1 | 8/8 (100) | 6.2-8.1 | 6.9 | 6/8 (75) | 0-4.7 | 2.4 | 4/8 (50) | 0-4 | 1.7 | 2/8 | 0-3.7 | 1.7 |
W2 | 9/10 (90) | 0-1,120 | 194 | 10/10 (100) | 5.8-6.8 | 6.4 | 9/10 (90) | 0-3.7 | 2.6 | 8/10 (80) | 0-3.3 | 2.2 | 0/10 | 0 | 0 |
W3 | 10/10 (100) | 6-1,550 | 534 | 10/10 (100) | 6-7.8 | 7 | 10/10 (100) | 2.9-4.8 | 3.8 | 10/10 (100) | 2-4.5 | 3.4 | 0/10 | 0 | 0 |
W4 | 10/10 (100) | 16-1,732 | 529 | 10/10 (100) | 6.4-7.6 | 7 | 10/10 (100) | 3.2-4.6 | 3.9 | 10/10 (100) | 2.8-4.3 | 3.4 | 0/10 | 0 | 0 |
W5 | 10/10 (100) | 2-2,420 | 687 | 10/10 (100) | 5.5-6.9 | 6.3 | 7/10 (70) | 0-3.2 | 1.7 | 5/10 (50) | 0-3.1 | 1.2 | 0/10 | 0 | 0 |
W6 | 10/10 (100) | 3-1,990 | 389 | 10/10 (100) | 5.5-7 | 6.3 | 9/10 (90) | 0-4.3 | 2.8 | 6/10 (60) | 0-5.1 | 2 | 1/10 | 0-3.4 | 1.3 |
W7 | 7/7 (100) | 5-2,420 | 445 | 7/7 (100) | 5.7-7.8 | 7 | 6/7 (86) | 0-3.8 | 2.6 | 5/7 (71) | 0-4.4 | 2.4 | 2/7 | 0-5.1 | 2.8 |
W8 | 10/10 (100) | 17-980 | 160 | 10/10 (100) | 6.3-8.6 | 7.1 | 8/10 (80) | 0-4.6 | 2.8 | 7/10 (70) | 0-4.4 | 2.3 | 0/10 | 0 | 0 |
14.
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Ana Roque Carmen Lopez-Joven Beatriz Lacuesta Laurence Elandaloussi Sariqa Wagley M. Dolores Furones Imanol Ruiz-Zarzuela Ignacio de Blas Rachel Rangdale Bruno Gomez-Gil 《Applied and environmental microbiology》2009,75(23):7574-7577
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).
Open in a separate windowaMg, Mytilus galloprovincialis; Cg, Crassostrea gigas; Rd, Ruditapes decussatus; Rp, R. phillipinarum; A, Alfacs; F, Fangar; ND, not determined; TCBS, thiosulfate citrate-bile salts-sucrose.Total DNA was extracted from each purified isolate using the Wizard genomic DNA purification kit (Promega), following the instructions of the manufacturer. A one-step PCR analysis was performed to identify/confirm which isolates were tl positive (species marker for V. parahaemolyticus). Further detection of the tdh or trh gene was carried out on all positive tl strains. All PCR analyses were carried out using the primers described by Bej et al. (2) with the following amplification conditions on the thermocycler (Eppendorf Mastercycler Personal): an initial denaturation at 95°C for 8 min, followed by 40 cycles of a 1-min denaturation at 94°C, annealing at 55°C for 1 min, elongation at 72° for 1 min, and a final extension of 10 min at 72°C. Positive and negative controls were included in all reaction mixtures: two positive controls, tl and tdh CAIM 1400 and trh CAIM 1772 (Collection of Aquatic Important Microorganisms [http://www.ciad.mx/caim/CAIM.html]), and negative control DNA-free molecular grade water (Sigma-Aldrich, Spain). Expected amplicons were visualized in 2% agarose gels stained with ethidium bromide.Fifty-eight isolates contained the gene tl in 2006 and 96 in 2008, which confirmed their identity as V. parahaemolyticus. In 2006, the distribution of the 58 isolates was as follows: 7 from 127 mussels, 34 from 180 oysters, and 17 from 30 R. decussatus clams. No tl-positive isolates were found in R. philippinarum. PCR analysis of the tl-positive isolates for the presence of the tdh or trh gene indicated that eight isolates contained the tdh gene and four contained the trh gene. In 2008, the source of the confirmed V. parahaemolyticus isolates was as follows: 31 from 88 oysters, 44 from 89 mussels, 9 from 30 R. decussatus clams, and 12 from 30 R. philippinarum clams. Of these, 17 were found to contain the tdh gene and 7 contained the trh gene. Two isolates (I806 and I1042) contained both toxigenic genes, tdh and trh.Putative tdh- and trh-positive PCR products were purified using the QIAquick PCR purification kit (Qiagen) following the manufacturer''s instructions and were sequenced bidirectionally by Macrogen Inc. Sequences were aligned using BioEdit (8) and analyzed using BLAST (National Center for Biotechnology Information). None of the toxigenic isolates was found positive by PCR analysis for the presence of open reading frame 8 of the phage 237 (16), a marker for the pandemic strain O3:K6.The isolates were fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) as described previously (3), and the resulting electrophoretic band patterns were analyzed with the GelCompar II software (v4.5; Applied Maths). The similarity matrix was calculated with the Jaccard coefficient with a band position tolerance of 0.8%, and the dendrogram was constructed with the Ward algorithm. A high level of genomic diversity was found among the 32 toxigenic isolates characterized by rep-PCR. Three clonal groups were identified (those having identical rep-PCR band patterns) (Fig. 1a to c).Open in a separate windowFIG. 1.rep-PCR dendrogram of toxigenic isolates of V. parahaemolyticus isolated in the Ebro delta. Letters denote clonal groups of isolates.In vitro antibiotic susceptibility tests were performed using the diffusion disc test following a previously described protocol (18). The antibiotics used were gentamicin (10 μg), oxolinic acid (10 μg), amoxicillin (25 μg), polymyxin B (300 UI), vancomycin (30 μg), trimethoprim sulfamethoxazole (1.25/23.75 μg), nitrofurantoin (300 μg), doxycyclin (30 μg), ceftazidime (30 μg), streptomycin (10 μg), neomycin (30 UI), penicillin (6 μg), flumequine (30 μg), tetracycline (30 μg), ampicillin (10 μg), kanamycin (30 μg), ciprofloxacin (5 μg), and sulfonamide (300 μg). All tests were performed in duplicate. A Student t test for two samples with unequal variance was performed to compare the sensitivity of all 2006 isolates against the sensitivity of 2008 isolates for each antibiotic (Microsoft Office Excel 97-2003). Antibiogram results revealed a lower susceptibility in 2008 than in 2006, indicating a possible shift in overall susceptibility. Results from the t test indicated that significantly lower susceptibility in 2008 was detected (P ≤ 0.05; n = 36) for the following antibiotics: vancomycin, polymyxin B, ampicillin, amoxicillin, gentamicin, neomycin, trimethoprim sulfamethoxazole, nitrofurantoin, doxycyclin, ceftazidime, tetracycline, flumequine, and ciprofloxacin.The serological types for 27 strains were determined by the agglutination method using commercially available V. parahaemolyticus antisera (Denka Seiken Ltd.; Cosmos Biomedical Ltd, United Kingdom) following the manufacturer''s instructions. Potentially toxigenic V. parahaemolyticus isolates collected in 2006 were serologically heterogeneous (8 out of the 11 isolates) (Table (Table1).1). In isolates collected in 2008, results were more homogenous, with seven serotypes found among 19 isolates analyzed. The O3:K6 serotype was not detected in any of the strains analyzed, in agreement with the open reading frame 8 PCR results.The present study is the first to report the detection of potentially diarrheal V. parahaemolyticus strains isolated from cultured bivalves on Spanish Mediterranean coasts, providing data on the presence of both tdh- and trh-positive isolates. V. parahaemolyticus has previously been detected in several European countries (4, 13, 21, 22). A recent study carried out in Spain detected tdh-positive V. parahaemolyticus strains from patients who had consumed fresh oysters in a market in Galicia on the Atlantic coast of Spain (12) and potentially pathogenic V. parahaemolyticus strains have also been reported in France (17). These studies indicate that the risk of infections caused by V. parahaemolyticus in Europe is low compared to that in America or Asia (15). However, this risk could have been underestimated, since V. parahaemolyticus is not included in the current European surveillance programs, such as the European Network for Epidemiological Surveillance and Control of Communicable Diseases.Toxigenic V. parahaemolyticus strains detected in this study were genomically and serologically heterogeneous. The pandemic serotype O3:K6 was not detected, and although attempts to isolate O3:K6 from the environment and from seafood have not always been successful in previous studies reviewed by Nair and coauthors (15), this finding seems to be in agreement with the fact that no outbreak of diarrhea was observed in the area. Interestingly, isolates I806 and I1042 have been found positive for both tdh and trh in PCR tests. The coexistence of tdh and trh genes has already been reported in isolates from Japan, the United States, and Mexico (3, 6, 11, 19, 23). To our knowledge, no occurrence of an environmental isolate positive for both tdh and trh had previously been reported in Europe. All isolates tested were slightly different in their antibiotic resistance profiles. Typically, a high level of resistance could be determined. The detection of tdh- and/or trh-positive V. parahaemolyticus strains for the first time on the Mediterranean coast emphasizes the need to monitor for the presence of potentially diarrheal vibrios and bacterial gastroenteritis, and these data should be taken into consideration to revise the European legislation on the requirements for shellfish harvested for consumption in order to include the surveillance of these pathogens in Europe. 相似文献
TABLE 1.
Vibrio parahaemolyticus isolates, serotypes, and origins and total number of vibrios/heterotrophic bacteria contained in the bivalveaIsolate | Date of collection | Organism and site of origin | Temp (°C) | Salinity (‰) | Gene(s) | Serotype | Bacterial count using indicated medium (CFU ml−1) | |
---|---|---|---|---|---|---|---|---|
TCBS agar | Marine agar | |||||||
I745 | 8 August 2006 | Mg-F | 24.5 | 37 | tdh | ND | 1.5 × 104 | 1.2 × 104 |
I793 | 14 August 2006 | Cg-A | 25 | 35 | tdh | ND | 9.2 × 102 | 8.5 × 103 |
I805 | 14 August 2006 | Cg-A | 25 | 35 | tdh | O2:KUT | 7.2 × 102 | 9 × 103 |
I806 | 14 August 2006 | Cg-A | 25 | 35 | tdh and trh | O3:K33 | 1.9 × 103 | 4.6 × 103 |
I809 | 14 August 2006 | Cg-A | 25 | 35 | tdh | O2:K28 | 8 × 104 | 7.3 × 102 |
I678 | 4 July 2006 | Rd-A | 28.6 | 36 | tdh | O2:K28 | 3.1 × 105 | 2.5 × 105 |
I628 | 4 July 2006 | Rd-A | 28.6 | 36 | tdh | O4:KUT | 2.9 × 104 | 8.4 × 104 |
I775 | 8 August 2006 | Cg-A | 24.5 | 37 | tdh | ND | 4.21 × 103 | 1.1 × 104 |
I691 | 4 July 2006 | Rd-A | 28.6 | 36 | trh | O1:K32 | 2.2 × 105 | 2.6 × 105 |
I712 | 27 July 2006 | Mg-A | 29.4 | 35.5 | trh | O1:KUT | 8.6 × 103 | 8.4 × 103 |
I765 | 8 August 2006 | Cg-F | 24.5 | 37 | trh | O4:K34 | 1 × 104 | Uncountable |
I980 | 22 July 2008 | Cg-A | 26.7 | 33.5 | tdh | O1:K32 | 2.7 × 104 | 1.3 × 104 |
I981 | 22 July 2008 | Cg-A | 26.7 | 33.5 | trh | O1:KUT | 1 × 104 | 2.2 × 104 |
I993 | 22 July 2008 | Cg-A | 26.7 | 33.5 | tdh | O5:K17 | 3 × 103 | 1.1 × 104 |
I994 | 29 July 2008 | Mg-A | 27.7 | 37 | trh | O3:KUT | 3.4 × 103 | 7 × 103 |
I1031 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh | O5:KUT | 5.5 × 104 | 3.3 × 104 |
I1034 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh | O3:KUT | 8.7 × 104 | 4 × 104 |
I1040 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh | O3:KUT | 1.6 × 104 | 3.2 × 104 |
I1042 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh and trh | ND | 2.8 ×104 | 3 × 104 |
I1050 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh | O1:KUT | 4.7 × 104 | 7.3 × 104 |
I1063 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O3:KUT | 7.9 ×104 | 1.4 × 104 |
I1065 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O2:KUT | 2.2 × 103 | 1.2 × 104 |
I1068 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O5:KUT | 2.6 × 104 | 5.2 × 104 |
I1069 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O3:KUT | 2.4 × 103 | 5.3 × 104 |
I1073 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O5:KUT | 2.3 × 103 | 7.5 × 103 |
I1074 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O3:KUT | 7.6 × 104 | 6.9 × 104 |
I1077 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O4:KUT | 1.7 × 103 | 1.6 × 103 |
I1079 | 20 August 2008 | Mg-F | 25.9 | 36 | trh | O3:KUT | 2.5 × 103 | 1.1 × 104 |
I1092 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | ND | 1.7 × 103 | 1.6 × 103 |
I1130 | 25 August 2008 | Rd-A | 26.4 | 35 | tdh | ND | 1.7 × 104 | 3.8 × 104 |
I1143 | 25 August 2008 | Rd-A | 26.4 | 35 | tdh | ND | 1.1 × 104 | 1.9 × 104 |
I1165 | 25 August 2008 | Rd-A | 26.4 | 35 | trh | O2:KUT | 4.4 × 104 | 6.8 × 104 |
I1133 | 25 August 2008 | Rp-F | 25.5 | 36.5 | tdh | ND | 3.4 × 104 | 4 × 104 |
I1134 | 25 August 2008 | Rp-F | 25.5 | 36.5 | tdh | ND | 3.9 × 104 | 5.8 × 104 |
I1158 | 25 August 2008 | Rp-F | 25.5 | 36.5 | trh | O4:KUT | 6.6 × 104 | 4.7 × 104 |
I1161 | 25 August 2008 | Rp-F | 25.5 | 36.5 | trh | O3:KUT | 2.2 × 104 | 6.6 × 104 |
17.
L. Pilloni P. Bianco C. Manieli G. Senes P. Coni L. Atzori N. Aste G. Faa 《European journal of histochemistry : EJH》2009,53(2)
Basal cell carcinoma (BCC) is a very common malignant skin tumor that rarely metastatizes, but is often locally aggressive. Several factors, like large size (more than 3 cm), exposure to ultraviolet rays, histological variants, level of infiltration and perineural or perivascular invasion, are associated with a more aggressive clinical course. These morphological features seem to be more determinant in mideface localized BCC, which frequently show a significantly higher recurrence rate. An immunohistochemical profile, characterized by reactivity of tumor cells for p53, Ki67 and alpha-SMA has been associated with a more aggressive behaviour in large BCCs. The aim of this study was to verify if also little (<3 cm) basal cell carcinomas can express immunohistochemical markers typical for an aggressive behaviour.Basal cell carcinoma (BCC) is a very common malignant skin tumor that rarely metastatizes, even If Is often locally aggressive. Several factors, like large size (more than 3 cm), face localization, exposure to ultraviolet rays, histological variants, infiltration level and perineural or perivascular invasion, are associated with a more aggressive clinical course. In particular, the incidence of metastasis and/or death correlates with tumors greater than 3 cm in diameter in which setting patients are said to have 1–2 % risk of metastases that increases to 20–25% in lesions greater than 5 cm and to 50% in lesions greater than 10 cm in diameter (Snow et al., 1994). Histologically morpheiform, keratotic types and infiltrative growth of BCC are also considered features of the most aggressive course (Crowson, 2006). This can be explained by the fact that both the superficial and nodular variants of BCC are surrounded by a continuous basement membrane zone comprising collagens type IV and V admixed with laminin, while the aggressive growth variants (i.e. morpheiform, metatypical, and infiltrative growth subtypes) manifest the absence of basement membrane (Barsky et al., 1987).The molecular markers which characterize aggressive BCC include: increased expression of stromolysin (MMP-3) and collagenase-1 (MMP-1) (Cribier et al., 2001), decreased expression of syndecan-1 proteoglycan (Bayer-Garner et al., 2000) and of anti-apoptotic protein bcl-2 (Ramdial et al., 2000; Staibano et al., 2001).C-ras , c-fos (Urabe et al., 1994; Van der Schroeff et al., 1990) and p53 tumor supressor gene mutations (Auepemikiate et al., 2002) are indicative of an aggressive course.Focusing upon bcl-2 and p53 expression in BCC, there have been numerous studies documenting the utility of bcl-2 as a marker of favourable clinical behaviour while p53 expression may be a feature of a more aggressive outcome (Ramdial et al., 2000; Staibano et al., 2001; Bozdogan et al., 2002).An increased expression of cytoskeletal microfilaments like α–smooth muscle actin, frequently found in invasive BCC subtypes (Jones JCR et al., 1989), may explain an enhanced tumor mobility and deep tissue invasion through the stroma. (Cristian et al., 2001; Law et al., 2003). The aim of this preliminary study was to verify if also little (<3 cm) basal cell carcinomas may express aggressive immunohistochemical markers like p53, Ki67 and alpha-SMA. We used 31 excisional BCCs with tumor size less than 2 cm (ranging from 2 up to 20 mm) and with different skin localization (19 in the face, 6 in the trunk and 6 in the body extremities). All cases were immunostained for p53, BCL2, Ki67 and alpha-smooth muscle actin (α-SMA) (Age Sex Location Hystotype Max.Dim Depth Ulc Ess Inf p53 Bcl-2 Ki67 AML 1 61 M Extr Keratotic 10×8 1 No +++ URD +++ + + - 2 61 M Face Adenoid 10×9 4 No + URD +++ - - - 3 64 M Extr Sup mult 11×13 0.8 No + DRD + - - - 4 73 M Face Nodular 10×8 2 Yes + DRD +++ + ++ +++ 5 84 M Face Nodular 9×12 2 Yes + DRD - - - - 6 84 M Face Adenoid 5 0.8 No + URD +++ - - - 7 84 M Extr Nodular 13×10 3 No + DRD +++ + + - 8 52 F Face Nodular 4 0.8 No + URD + + + - 9 76 F Face Adenoid 10×4 4 No + DRD +++ - ++ - 10 77 F Face Morph 8×6 1 Yes +++ DRD +++ - - - 11 86 M Face Morph 8 1 Yes + DRD +++ - + + 12 63 F Face Adenoid 4 1 No + URD ++ + + + 13 76 F Face Nodular 7 1.5 No + DRD +++ + ++ - 14 84 M Face Nodular 11 4 Yes +++ DRD + - - + 15 63 F Face Keratotic 10×6 1.8 No ++ DRD - + ++ - 16 68 F Trunk Sup mult 10×6 0.7 No ++ URD + + - - 17 67 M Face Sup mult 12×6 0.4 No + URD + - + - 18 67 M Extr Sup mult 4×3 0.3 No + URD + +++ + - 19 32 F Extr Sup mult 1×3 0.4 No + URD + + + - 20 45 M Trunk Nodular 7×5 2 Yes +++ URD + + + - 21 62 M Trunk Sup mult 11×7 0.9 No ++ URD - ++ - ++ 22 65 M Trunk Adenoid 7×6 1.5 No + URD +++ + + - 23 72 M Trunk Nodular 12×6 1 No + URD +++ - + + 24 86 F Face Keratotic 20×11 3.1 No ++ DRD + + + - 25 85 M Face Nodular 0.5 1.3 No ++ DRD ++ + + - 26 74 F Extr Nodular 4×4 0.9 No + URD - - + - 27 71 M Face Nodular 6×12 1.7 No + DRD - - + - 28 64 F Trunk Sup mult 1.3×1.5 0.4 No ++ URD +++ - - - 29 78 F Face Nodular 4×3 1.5 No ++ DRD ++ + - +++ 30 80 M Face Keratotic 4×4 1.6 Yes + DRD - - + +++