Development and Application of a New Method for Specific and Sensitive Enumeration of Spores of Nonproteolytic Clostridium botulinum Types B,E, and F in Foods and Food Materials

The highly potent botulinum neurotoxins are responsible for botulism, a severe neuroparalytic disease. Strains of nonproteolytic Clostridium botulinum form neurotoxins of types B, E, and F and are the main hazard associated with minimally heated refrigerated foods. Recent developments in quantitative microbiological risk assessment (QMRA) and food safety objectives (FSO) have made food safety more quantitative and include, as inputs, probability distributions for the contamination of food materials and foods. A new method that combines a selective enrichment culture with multiplex PCR has been developed and validated to enumerate specifically the spores of nonproteolytic C. botulinum. Key features of this new method include the following: (i) it is specific for nonproteolytic C. botulinum (and does not detect proteolytic C. botulinum), (ii) the detection limit has been determined for each food tested (using carefully structured control samples), and (iii) a low detection limit has been achieved by the use of selective enrichment and large test samples. The method has been used to enumerate spores of nonproteolytic C. botulinum in 637 samples of 19 food materials included in pasta-based minimally heated refrigerated foods and in 7 complete foods. A total of 32 samples (5 egg pastas and 27 scallops) contained spores of nonproteolytic C. botulinum type B or F. The majority of samples contained <100 spores/kg, but one sample of scallops contained 444 spores/kg. Nonproteolytic C. botulinum type E was not detected. Importantly, for QMRA and FSO, the construction of probability distributions will enable the frequency of packs containing particular levels of contamination to be determined.Food-borne botulism is a severe and deadly intoxication caused by the consumption of food containing as little as 30 to 100 ng of preformed botulinum neurotoxin (45). More than 2,500 cases of botulism were reported in Europe in 1999 and 2000, with the majority of cases in the east of the continent (44). Currently, 25 to 50 food-borne botulism cases are diagnosed annually in the United States (27). There are seven distinct botulinum neurotoxins (types A to G) and a number of subtypes (6, 26, 45). In view of the potency of the botulinum neurotoxin and the severity of botulism, four phylogenetically distinct bacteria are grouped together as the Clostridium botulinum species, solely on the basis of their ability to form botulinum neurotoxin. The divergence between these four distinct bacteria is strong enough to merit their classification as distinct species and in some cases is significantly greater than that between bacteria belonging to different genera, e.g., Bacillus subtilis and Staphylococcus aureus (7). Two of these bacteria (proteolytic C. botulinum and nonproteolytic C. botulinum) are responsible for the majority of cases of food-borne botulism. Strains of proteolytic C. botulinum produce neurotoxins of type A, B, or F, form spores of high heat resistance, and have a minimum growth temperature of approximately 12°C (39). Strains of nonproteolytic C. botulinum produce neurotoxins of type B, E, or F, form spores of moderate heat resistance, and are able to grow and form toxin at 3°C (18, 48) and are recognized as the major hazard associated with minimally heated refrigerated foods (4, 37, 43, 44, 48). These new foods meet consumer demand for high-quality, convenient foods that are low in preservatives, and sales are presently increasing by about 10% per annum in many countries (3, 47).Quantitative microbiological risk assessment (QMRA) is now established as an important microbiology food safety tool (42). Process risk models have been used to assess the safety of specific foods with respect to nonproteolytic C. botulinum and the food-borne botulism hazard (e.g., 2, 41). These process risk models benefit from high-quality information, including that on the incidence of spores of nonproteolytic C. botulinum spores in food materials. The implementation of food safety objectives (FSO) also benefits from the availability of high-quality information on the microbial contamination of foods and food materials (24). This information is most effective in the form of probability distributions rather than as average spore concentrations or other statistics.The difficulty with enumerating nonproteolytic C. botulinum in foods is that there is no effective selective culture medium available. Surveys of the extent of contamination of foods and food materials have used a nonselective enrichment followed by either testing for neurotoxin using a mouse test or enzyme-linked immunosorbent assay (ELISA) or testing for the presence of neurotoxin genes using a PCR test (3, 10, 13, 35, 38, 39). This approach, however, is not optimized for nonproteolytic C. botulinum or proteolytic C. botulinum (therefore potentially failing to recover all spores of either organism) and may also not distinguish nonproteolytic C. botulinum from proteolytic C. botulinum. Heating at 80°C for 10 min followed by incubation at 35°C (54) may be reasonably selective for proteolytic C. botulinum, but there is no similar approach for nonproteolytic C. botulinum, although incubation at 28°C (54) may offer an element of selection. It is necessary, therefore, to develop a method to enumerate spores of nonproteolytic C. botulinum in food materials that is robust and optimized, as well as sensitive and specific for this particular pathogen (and does not also detect proteolytic C. botulinum). When enumerating bacteria in foods, it is essential to demonstrate the efficiency of the method by verifying that small concentrations (in the present study, spores of nonproteolytic C. botulinum) can be detected following addition to test samples.This paper describes the development, validation, and application of a new method to enumerate spores of nonproteolytic C. botulinum in foods and in food materials. This method has been designed to generate data for the construction of probability distributions that can be used in QMRA and FSO settings. Most of the effort has been dedicated to the development and evaluation of the enrichment procedure rather than the PCR test, as the PCR test has received much attention from others (e.g., 3, 10, 16, 36, 38). A low-temperature selective-enrichment procedure is described that has been optimized specifically for nonproteolytic C. botulinum over proteolytic C. botulinum and other bacteria. In order to detect low concentrations of spores, large quantities (200 g) of food materials and foods have been tested. Specific detection of neurotoxin genes is achieved by the use of an established multiplex PCR (36), with an internal amplification control now included (25). By the use of a set of control samples inoculated with defined concentrations of spores of nonproteolytic C. botulinum, the detection limit has been estimated for each food material and food tested. The method has been used in an extensive survey of raw materials intended for use in pasta ready meals, as well as the final meals themselves. The implications for risk assessment and risk management of chilled foods are discussed.     

Comparative Genomic Hybridization Analysis of Two Predominant Nordic Group I (Proteolytic) Clostridium botulinum Type B Clusters

Comparative genomic hybridization analysis of 32 Nordic group I Clostridium botulinum type B strains isolated from various sources revealed two homogeneous clusters, clusters BI and BII. The type B strains differed from reference strain ATCC 3502 by 413 coding sequence (CDS) probes, sharing 88% of all the ATCC 3502 genes represented on the microarray. The two Nordic type B clusters differed from each other by their response to 145 CDS probes related mainly to transport and binding, adaptive mechanisms, fatty acid biosynthesis, the cell membranes, bacteriophages, and transposon-related elements. The most prominent differences between the two clusters were related to resistance to toxic compounds frequently found in the environment, such as arsenic and cadmium, reflecting different adaptive responses in the evolution of the two clusters. Other relatively variable CDS groups were related to surface structures and the gram-positive cell wall, suggesting that the two clusters possess different antigenic properties. All the type B strains carried CDSs putatively related to capsule formation, which may play a role in adaptation to different environmental and clinical niches. Sequencing showed that representative strains of the two type B clusters both carried subtype B2 neurotoxin genes. As many of the type B strains studied have been isolated from foods or associated with botulism, it is expected that the two group I C. botulinum type B clusters present a public health hazard in Nordic countries. Knowing the genetic and physiological markers of these clusters will assist in targeting control measures against these pathogens.Clostridium botulinum produces a potent neurotoxin during its growth. The toxin causes a potentially lethal paralytic disease, botulism, in humans and animals. The classical food-borne botulism follows the consumption of toxin-containing food or drink, while infant and adult intestinal botulism results from in vivo spore germination, outgrowth, and toxin production in the gut. Apart from attenuated intestinal microbial population, other factors affecting the colonization of C. botulinum in the intestinal forms of botulism are not known.Based on their physiology and genetic background, C. botulinum strains are divided into groups I to IV (13). Strains of groups I and II are associated with human disease. Group I strains produce neurotoxin serotypes A, B, and/or F, while the group II strains produce type B, E, or F toxin. Physiologically, groups I and II differ markedly from each other as well as from groups III and IV. Genomic analysis of group I and II C. botulinum strains by 16S rrn sequencing (13), ribotyping (10), and amplified fragment length polymorphism (11, 15, 16) is consistent with the divergent physiologies of the two groups (18).Nordic C. botulinum group I strains show a remarkable homogeneity (15, 20, 21, 23). In a large pulsed-field gel electrophoresis (PFGE) analysis, the majority of group I strains isolated from various sources from Finland, Norway, and Denmark formed type B neurotoxin and clustered into two large groups, with the members of each group sharing identical or nearly identical restriction patterns (20, 23). Many of these strains were recovered from honey for human consumption (23), and one strain was related to an infant botulism case (22). Apart from a recent study showing that strains of the two type B clusters, further referred to as clusters BI and BII, differ in their abilities to grow at extreme temperatures (12), the physiological, epidemiological, and genetic markers of the two clusters are not known. An understanding of such traits will assist in designing control measures against these potential food- and environment-borne pathogens.The availability of group I C. botulinum genome sequences has enabled the construction of whole-genome DNA microarrays and a comprehensive genomic analysis of C. botulinum strains (26, 27). In this paper, we describe a comparative genomic hybridization (CGH) analysis of 32 Nordic group I C. botulinum type B cluster BI or BII strains with a DNA microarray based on the protein-coding sequences (CDS) in the ATCC 3502 genome. Strains within each cluster showed no substantial variation. Furthermore, strains belonging to the two clusters differed by their responses to 145 CDS probes, suggesting differential resistance to toxic compounds and a relatively large antigenic variability. Sequencing of botB in a representative cluster BI strain and a representative cluster BII strain revealed subtype B2 neurotoxin genes in both strains.     

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10³ and 10⁵ CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.Botulinum neurotoxins (BoNTs), the causative agents of botulism, are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven serotypes, A to G. While the botulinum neurotoxins BoNT/A, BoNT/B, BoNT/E, and BoNT/F are known to cause botulism in humans, BoNT/C and BoNT/D are frequently associated with botulism in cattle and birds. Despite its toxicity, BoNT/G has not yet been linked to naturally occurring botulism (26).Botulism is a life-threatening illness caused by food contaminated with BoNT (food-borne botulism), by the uptake and growth of C. botulinum in wounds (wound botulism), or by colonization of the intestinal tract (infant botulism) (14). In addition, C. botulinum and the botulinum neurotoxins are regarded as potential biological warfare agents (8).The gold standard for the detection of BoNTs from food or clinical samples is still the mouse lethality assay, which is highly sensitive but rather time-consuming. In addition to various immunological assays for BoNT detection, several conventional and real-time PCR-based assays for the individual detection of bont genes have been reported (2, 9-12, 15, 20, 23, 27-30). A major improvement is the simultaneous detection of more than one serotype, which results in a reduction of effort and in the materials used. In recent years, both conventional and real-time PCR-based multiplex assays have been developed for the simultaneous detection of C. botulinum serotypes (1, 6, 22, 24). To date, however, no internally controlled multiplex real-time PCR assay for the simultaneous detection and differentiation of all four serotypes relevant for humans has been reported.We describe here a highly specific and sensitive multiplex real-time PCR assay based on the 5′-nuclease TaqMan chemistry (17) for the simultaneous detection of the C. botulinum types A, B, E, and F, including an internal amplification control (IAC). Furthermore, we developed six different singleplex assays based on the TaqMan chemistry for the detection of C. botulinum serotypes A to F. Assays were validated on 42 C. botulinum strains, 57 non-C. botulinum strains, on spiked food samples, and on real samples from cases of botulism in Germany.     

Sequence Diversity of Genes Encoding Botulinum Neurotoxin Type F

Botulism due to type F botulinum neurotoxin (BoNT/F) is rare (<1% of cases), and only a limited number of clostridial strains producing this toxin type have been isolated. As a result, analysis of the diversity of genes encoding BoNT/F has been challenging. In this study, the entire bont/F nucleotide sequences were determined from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak investigations. We examined proteolytic and nonproteolytic Clostridium botulinum type F strains, bivalent strains, including Bf and Af, and Clostridium baratii type F strains. Phylogenetic analysis revealed that the bont/F genes examined formed 7 subtypes (F1 to F7) and that the nucleotide sequence identities of these subtypes differed by up to 25%. The genes from proteolytic (group I) C. botulinum strains formed subtypes F1 through F5, while the genes from nonproteolytic (group II) C. botulinum strains formed subtype F6. Subtype F7 was composed exclusively of bont/F genes from C. baratii strains. The region of the bont/F5 gene encoding the neurotoxin light chain was found to be highly divergent compared to the other subtypes. Although the bont/F5 nucleotide sequences were found to be identical in strains harboring this gene, the gene located directly upstream (ntnh/F) demonstrated sequence variation among representative strains of this subtype. These results demonstrate that extensive nucleotide diversity exists among genes encoding type F neurotoxins from strains with different phylogenetic backgrounds and from various geographical sources.Botulism is a potentially fatal disease caused solely by the action of serologically distinct neurotoxins (BoNT/A, -B, -C, -D, -E, -F, or -G) which prevent acetylcholine release at neuromuscular junctions, resulting in paralysis. Food-borne botulism may result from the ingestion of a preformed toxin that is produced in inadequately preserved food. Under certain conditions, botulinum neurotoxin-producing Clostridium sp. may colonize and produce toxin in wounds (wound botulism) or in the intestine (infant botulism or adult colonization). Globally, human botulism cases are associated with botulinum neurotoxin serotypes A, B, E, and rarely F. The Centers for Disease Control and Prevention (CDC) maintains active surveillance for botulism cases in the United States. Of 1,269 U.S. cases of botulism reported to the CDC between 1981 and 2002, approximately 1% were due to type F toxin (13). An additional 10 cases of type F botulism were reported to the CDC from 2003 to 2007 (http://www.cdc.gov/nationalsurveillance/botulism_surveillance.html).Type F botulism was first described in 1960 following an outbreak occurring in Denmark involving liver paste (30). The organism isolated in this outbreak metabolically resembled proteolytic Clostridium botulinum strains of types A and B. In a subsequent outbreak, type F toxin was found to be produced by a nonproteolytic C. botulinum strain isolated from venison jerky (29). Bivalent toxin-producing strains have been described, including Bf strains isolated from infants in the United States and England (1, 16, 17, 35) and an Af strain isolated from individuals in Argentina with food-borne botulism (11). Bivalent strains may produce higher titers of one toxin type, which are denoted with a capital letter. The only reported organism isolated from infants with botulism due to type F toxin alone (i.e., not associated with additional serotypes as in bivalent strains) is Clostridium baratii (2, 14, 24). In addition, C. baratii type F has been isolated from adults with botulism (28) as well as suspect foods associated with botulism cases (15; CDC, unpublished data).Botulinum neurotoxin genes (bont) are typically found within toxin gene clusters that include other genes encoding components of the toxin complex (ha70, ha17, ha33, ntnh), regulatory proteins (botR), or proteins with unknown functions (p47, orfX1, orfX2, orfX3). Two general toxin gene cluster arrangements have been described, including the orfX cluster (orfX3-orfX2-orfX1-botR-p47-ntnh-bont) and the ha cluster (ha70-ha17-ha33-botR-ntnh-bont) (21, 22). The bont/F genes of type F and type Bf strains examined by Hill et al. (21) were found in an orfX cluster.The amino acid sequence identities of the BoNT serotypes A to G range from approximately 35 to 70% (36). In addition, within nearly all toxin serotypes, various levels of amino acid sequence variation have been observed, resulting in the identification of toxin subtypes (20, 36, 37).Although a limited number of genes encoding type F botulinum neurotoxin have been sequenced, a comparison of sequences available in public databases indicates that significant diversity exists within this serotype. The nucleotide sequence identity of the type F neurotoxin gene from the proteolytic strain Langeland differs from that of the gene in the nonproteolytic strain 202F by 7%. The type F gene from C. baratii strain ATCC 43756 differs from those of Langeland and 202F by 18% and 20%, respectively. Although the bivalent (Bf) strain CDC3281 is phenotypically proteolytic, the toxin gene shows greater similarity to those from nonproteolytic strains (34). In addition to metabolic differences observed between proteolytic and nonproteolytic C. botulinum strains as well as C. baratii, these organisms are phylogenetically distinct based on differences among their 16S rRNA sequences (5, 20).In order to define the degree of genetic diversity among strains encoding botulinum neurotoxin type F, we sequenced the bont/F gene and partially characterized the toxin gene cluster by using a panel of 33 strains with diverse origins. These strains were selected from those available in the CDC culture collection as well as several isolated in Argentina. The only reported Af strains have been isolated in Argentina. Among 68 outbreaks of serotype-confirmed food-borne botulism in Argentina between 1922 and 2007, type F was isolated in two outbreaks, and type Af was isolated in one outbreak. In addition, Lúquez et al. (26) reported isolation of type F and Af strains from Argentine soils.Here, we report that analysis of the bont/F genes from the strains examined in this study revealed a high degree of nucleotide sequence heterogeneity and the identification of seven type F subtypes (F1 to F7). In addition, the nucleotide sequence of one subtype (F5) has not been previously reported and contains evidence of recombination compared to the other subtypes.     

Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical,Food, and Environmental Samples

Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.Botulinum neurotoxins (BoNTs) are the most toxic agents known, and as little as 30 ng neurotoxin is potentially lethal to humans (36). These toxins are responsible for botulism, a disease characterized by flaccid paralysis. Seven antigenically distinct BoNTs are known (types A to G), and BoNT types A, B, E, and F are the principal types associated with human botulism (37). Significant sequence diversity and antigenically variable subtypes have recently been reported for the type A, B, and E neurotoxin genes (14, 22, 23, 42).Apart from the species Clostridium botulinum, which itself consists of four phylogenetically distinct groups of organisms, some strains of other clostridia, namely Clostridium butyricum and Clostridium baratii, are also known to produce BoNTs (2, 4, 7, 13, 20, 26, 34, 44). Also, strains that produce two toxins and strains carrying silent toxin genes have been reported (8, 22, 24, 39). Due to the great physiological variation of the BoNT-producing clostridia, their isolation and identification cannot depend solely on biochemical characteristics (32). Indeed, the standard culture methods take into consideration only C. botulinum and not C. baratii and C. butyricum, and identification and confirmation require detection of BoNT by a standard mouse bioassay (SMB) (12). The SMB is highly sensitive and specific but also expensive, time-consuming, and undesirable because of the use of experimental animals. Detection of neurotoxin gene fragments by PCR is a rapid alternative method for detection and typing of BoNT-producing clostridia (3). Different PCR methods have been described for detecting neurotoxin type A-, B-, E-, and F-producing clostridia (9, 15-18, 21, 40, 41).A previously described multiplex PCR method able to simultaneously detect type A, B, E, and F neurotoxin genes is a useful tool for rapid detection of the BoNT-producing clostridia (31). While this method generally has a high level of inclusivity for detection of type B, E, and F neurotoxin genes, limitations for detection of the recently described subtype A2, A3, and A4 strains have been identified (6, 28). To increase the efficiency of this multiplex PCR method, new primers were designed to detect genes for all identified type A neurotoxin subtypes (19). Additionally, an internal amplification control (IAC) was added according to ISO 22174/2005. The specificity and selectivity of this multiplex PCR method were evaluated in comparison with an SMB (12) using target and nontarget strains, and the robustness was assessed using clinical, food, and environmental samples. Moreover, to evaluate the applicability of this multiplex PCR method, a survey with food and environmental samples was performed in a German food control laboratory.     

Expression of the Clostridium botulinum A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex

Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most potent toxins known in nature and are characterized as category A select agents since they are considered potential bioterrorism threats (3). BoNTs can be distinguished immunologically into seven serotypes by using homologous antitoxins, designated A to G. BoNT/A is of particular interest, since it is frequently implicated in cases of botulism and is a significant threat in bioterrorism (1, 10).BoNT is a 150-kDa protein composed of a heavy chain (100 kDa) and a light chain (50 kDa) linked by a disulfide bond and noncovalent molecular interactions (24). The heavy chain (H) has two functional domains, a transmembrane domain and a receptor binding domain. The light chain (L) is a zinc-dependent protease which specifically cleaves one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors, resulting in the blockage of evoked acetylcholine release at the skeletal neuromuscular junction (8).Previous studies have found that the bont genes of all strains of C. botulinum and neurotoxigenic strains of Clostridium butyricum and Clostridium baratii have a set of genes located upstream of the bont and ntnh genes that are organized as gene clusters (5, 7, 23). The two known primary types of clusters are (i) a hemagglutinin (ha) cluster and (ii) an orfX cluster with open reading frames (ORFs) of unknown functions. The ha cluster consists of genes encoding HA17, HA33, HA70, BotR, and NTNH. The orfX cluster consists of genes encoding ORFX3, ORFX2, ORFX1, P47, P21, and NTNH. Previous studies indicate that BoNT/A subtypes possess either a ha cluster or an orfX cluster associated with their expressed bont gene, depending on the subtype and strain (5, 11, 13-15, 33).It has been shown that the BoNT complex can form stable toxin complexes (TCs) of various sizes, including LL-TC (∼900 kDa), L-TC (∼500 kDa), and M-TC (∼300 kDa) composed of various combinations of HA proteins, NTNH, and BoNT (19, 21, 23, 29, 31, 34). M-TC contains BoNT and NTNH but has no HA proteins, whereas LL-TC and L-TC contain different ratios of the BoNT, NTNH, and HA proteins (21, 22, 29, 34). The biological and structural roles of the complex proteins are not completely characterized, although it has been proposed that they serve the role of protecting BoNT from harsh conditions, including pH, salt, temperature, and digestive enzymes, and that they assist BoNT translocation across the intestinal epithelial layer (2, 6, 17). A recent report indicated that the nontoxic proteins serve as adjuvants and contribute to the immunogenicity of BoNT/A (25).The production of botulinum TCs is known to vary with different serotypes and strains, medium composition, and culture conditions (21, 24, 31). The LL-TC has only been observed in proteolytic strains (group I). Serotype A to D strains produce M-TC and L-TC in their culture medium, while serotype E and F strains produce only M-TC (17, 18).In 1986, a Japanese group isolated four HA-negative C. botulinum strains from infant botulism cases that produced only M-TC (300 kDa). They assigned the strains to subtype A2 (14, 30). In 2004, our laboratory confirmed on a genomic level that the BoNT/A2 subtype contained the orfX cluster instead of the ha cluster (12). Since then, more arrangements and combinations of neurotoxin gene clusters were characterized along with more BoNT subtypes (13, 20, 33). However, the function of the orfX genes and the role of the presumptive protein products and their role in the TCs are still unknown, including whether ORFX proteins can form a TC with the expressed toxin analogous to the ha cluster proteins.In this study, the BoNT/A2 TC was purified from a native culture to determine if the orfX cluster proteins remain associated with BoNT/A2. To better understand the role of the orfX cluster genes, the orfX cluster proteins of C. botulinum A2 strains (ORFX1, ORFX3, P47, and the middle part of NTNH) was expressed using either an Escherichia coli or a C. botulinum expression system in this study. Antibodies against individual expressed orfX cluster proteins were then raised by immunizing a rabbit and mice. These antibodies were then used as probes to investigate the expression pattern of the orfX cluster genes in the native A2 culture. ORFX2, which could not be expressed, was detected by N-terminal protein sequencing.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Development of a Two-Color Fluorescence In Situ Hybridization Technique for Species-Level Identification of Human-Infectious Cryptosporidium spp.

A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification of Cryptosporidium parvum and C. hominis was developed. The assay is a simple, rapid, and cost-effective tool for the detection of the major Cryptosporidium species of concern to public health.Cryptosporidium (Apicomplexa) is a genus of protozoan parasites with species and genotypes that infect humans, domesticated livestock, companion animals, and wildlife worldwide (5, 6, 14, 15, 20, 23). The majority of cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or C. hominis (8, 10, 19, 24), although rare cases due to species such as C. meleagridis, C. felis, or C. canis have been reported (8, 9, 11-13, 17, 18, 22). The specific identification and characterization of Cryptosporidium species are central to the control of this disease in humans and a wide range of animals.One of the most widely adopted techniques for the identification of microorganisms in complex microbial communities is fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes (2-4). This method relies on the hybridization of synthetic oligonucleotide probes to specific regions within the rRNA of the organism. While FISH has been applied for the detection of Cryptosporidium oocysts in water samples (21), no FISH probes that successfully differentiate C. hominis from C. parvum have been reported.We have reported previously on the design of a species-specific probe, Cpar677, that detects C. parvum (1). In this study, we report on the design and validation of a C. hominis species-specific probe, Chom253. Together, the two probes were used here for the development of a two-color, microscopy-based FISH assay for the simultaneous detection of C. parvum and C. hominis.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.