Chitinolytic activity of phylogenetically diverse Bacillus cereus sensu lato from natural environments

Bacillus cereus s.l., Gram-positive endospore-forming bacilli, persist ubiquitously in different natural habitats and play various ecological roles. Nevertheless, although chitin is one of the most abundant polymer on Earth, the study of the ability of B. cereus s.l. to hydrolyze this polymer were limited to individual B. cereus and B. thuringiensis strains only. Thus, to fill this gap in this research we focused on (i) the linkage between the capability to chitin degradation and the phylogenetic relatedness of B. cereus s.l. strains, and (ii) the genetic background of chitinolytic properties of these bacilli. Our results showed that chitin degradation is common among the B. cereus group members, yet strains clustered into particular phylogenetic groups differ in their chitinolytic capacity. Separate clustering of chitinolytic and non-chitinolytic strains in the phylogenetic tree indicates the ecotypic structure of these isolates. Two proteins belonging to subfamily A (ChiA) and subfamily B (ChiB) of the glycoside hydrolase GH18 family exhibited simultaneous chitobiosidase and endochitinase activities, and are responsible for chitin utilization by environmental B. cereus s.l. isolates.     

Population structure and evolution of the Bacillus cereus group

Representative strains of the Bacillus cereus group of bacteria, including Bacillus anthracis (11 isolates), B. cereus (38 isolates), Bacillus mycoides (1 isolate), Bacillus thuringiensis (53 isolates from 17 serovars), and Bacillus weihenstephanensis (2 isolates) were assigned to 59 sequence types (STs) derived from the nucleotide sequences of seven alleles, glpF, gmk, ilvD, pta, pur, pycA, and tpi. Comparisons of the maximum likelihood (ML) tree of the concatenated sequences with individual gene trees showed more congruence than expected by chance, indicating a generally clonal structure to the population. The STs followed two major lines of descent. Clade 1 comprised B. anthracis strains, numerous B. cereus strains, and rare B. thuringiensis strains, while clade 2 included the majority of the B. thuringiensis strains together with some B. cereus strains. Other species were allocated to a third, heterogeneous clade. The ML trees and split decomposition analysis were used to assign STs to eight lineages within clades 1 and 2. These lineages were defined by bootstrap analysis and by a preponderance of fixed differences over shared polymorphisms among the STs. Lineages were named with reference to existing designations: Anthracis, Cereus I, Cereus II, Cereus III, Kurstaki, Sotto, Thuringiensis, and Tolworthi. Strains from some B. thuringiensis serovars were wholly or largely assigned to a single ST, for example, serovar aizawai isolates were assigned to ST-15, serovar kenyae isolates were assigned to ST-13, and serovar tolworthi isolates were assigned to ST-23, while other serovars, such as serovar canadensis, were genetically heterogeneous. We suggest a revision of the nomenclature in which the lineage and clone are recognized through name and ST designations in accordance with the clonal structure of the population.     

Phylogenetic Analysis Reveals Common Antimicrobial Resistant Campylobacter coli Population in Antimicrobial-Free (ABF) and Commercial Swine Systems

The objective of this study was to compare the population biology of antimicrobial resistant (AR) Campylobacter coli isolated from swine reared in the conventional and antimicrobial-free (ABF) swine production systems at farm, slaughter and environment. A total of 200 C. coli isolates selected from fecal, environmental, and carcass samples of ABF (n = 100) and conventional (n = 100) swine production systems were typed by multilocus sequence typing (MLST). Sequence data from seven housekeeping genes was analyzed for the identification of allelic profiles, sequence types (STs) and clonal complex determination. Phylogenetic trees were generated to establish the relationships between the genotyped isolates. A total of 51 STs were detected including two novel alleles (glnA 424 and glyA 464) and 14 novel STs reported for the first time. The majority of the C. coli isolates belonged to ST-854 (ABF: 31, conventional: 17), and were grouped in clonal complex ST-828 (ABF: 68%, conventional: 66%). The mean genetic diversity (H) for the ABF (0.3963+/−0.0806) and conventional (0.4655+/−0.0714) systems were similar. The index of association () for the ABF ( = 0.1513) and conventional ( = 0.0991) C. coli populations were close to linkage equilibrium, indicative of a freely recombining population. Identical STs were detected between the pigs and their environment both at farm and slaughter. A minimum spanning tree revealed the close clustering of C. coli STs that originated from swine and carcass with those from the environment. In conclusion, our study reveals a genotypic diverse C. coli population that shares a common ancestry in the conventional and ABF swine production systems. This could potentially explain the high prevalence of antimicrobial resistant C. coli in the ABF system in the absence of antimicrobial selection pressure.     

Evolution of pathogenicity in the Bacillus cereus group

The Bacillus cereus group of bacteria comprises soil-dwelling saprophytes but on occasion these bacteria can cause a wide range of diseases in humans, including food poisoning, systemic infections and highly lethal forms of anthrax. While anthrax is almost invariably caused by strains from a single evolutionary lineage, Bacillus anthracis, variation in the virulence properties of strains from other lineages has not been fully addressed. Using multi-locus sequence data from 667 strains, we reconstructed the evolutionary history of the B. cereus group in terms of both clonal inheritance and recombination. The strains included 155 clinical isolates representing B. anthracis, and isolates from emetic and diarrhoeal food poisoning, septicaemia and related infections, wound, and lung infections. We confirmed the existence of three major clades and found that clinical isolates of B. cereus (with the exception of emetic toxin-producing strains) are evenly distributed between and within clades 1 and 2. B. anthracis in particular and emetic toxin-producing B. cereus show more clonal structure and are restricted to clade 1. Our characterization of the patterns of genetic exchange showed that there exist partial barriers to gene flow between the three clades. The pathogenic strains do not exhibit atypically high or low rates of recombination, consistent with the opportunistic nature of most pathogenic infections. However, there have been a large number of recent imports in clade 1 of strains from external origins, which is indicative of an on-going shift in gene-flow boundaries for this clade.     

Multilocus Sequence Typing Scheme for Bacteria of the Bacillus cereus Group

In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.     

Distribution of Burkholderia pseudomallei in Northern Australia,a Land of Diversity

Burkholderia pseudomallei is a Gram-negative soil bacillus that is the etiological agent of melioidosis and a biothreat agent. Little is known about the biogeography of this bacterium in Australia, despite its hyperendemicity in the northern region of this continent. The population structure of 953 Australian B. pseudomallei strains representing 779 and 174 isolates of clinical and environmental origins, respectively, was analyzed using multilocus sequence typing (MLST). Bayesian population structure and network SplitsTree analyses were performed on concatenated MLST loci, and sequence type (ST) diversity and evenness were examined using Simpson''s and Pielou''s indices and a multivariate dissimilarity matrix. Bayesian analysis found two B. pseudomallei populations in Australia that were geographically distinct; isolates from the Northern Territory were grouped mainly into the first population, whereas the majority of isolates from Queensland were grouped in a second population. Differences in ST evenness were observed between sampling areas, confirming that B. pseudomallei is widespread and established across northern Australia, with a large number of fragmented habitats. ST analysis showed that B. pseudomallei populations diversified as the sampling area increased. This observation was in contrast to smaller sampling areas where a few STs predominated, suggesting that B. pseudomallei populations are ecologically established and not frequently dispersed. Interestingly, there was no identifiable ST bias between clinical and environmental isolates, suggesting the potential for all culturable B. pseudomallei isolates to cause disease. Our findings have important implications for understanding the ecology of B. pseudomallei in Australia and for potential source attribution of this bacterium in the event of unexpected cases of melioidosis.     

Cell wall carbohydrate compositions of strains from the Bacillus cereus group of species correlate with phylogenetic relatedness

Members of the Bacillus cereus group contain cell wall carbohydrates that vary in their glycosyl compositions. Recent multilocus sequence typing (MLST) refined the relatedness of B. cereus group members by separating them into clades and lineages. Based on MLST, we selected several B. anthracis, B. cereus, and B. thuringiensis strains and compared their cell wall carbohydrates. The cell walls of different B. anthracis strains (clade 1/Anthracis) were composed of glucose (Glc), galactose (Gal), N-acetyl mannosamine (ManNAc), and N-acetylglucosamine (GlcNAc). In contrast, the cell walls from clade 2 strains (B. cereus type strain ATCC 14579 and B. thuringiensis strains) lacked Gal and contained N-acetylgalactosamine (GalNAc). The B. cereus clade 1 strains had cell walls that were similar in composition to B. anthracis in that they all contained Gal. However, the cell walls from some clade 1 strains also contained GalNAc, which was not present in B. anthracis cell walls. Three recently identified clade 1 strains of B. cereus that caused severe pneumonia, i.e., strains 03BB102, 03BB87, and G9241, had cell wall compositions that closely resembled those of the B. anthracis strains. It was also observed that B. anthracis strains cell wall glycosyl compositions differed from one another in a plasmid-dependent manner. When plasmid pXO2 was absent, the ManNAc/Gal ratio decreased, while the Glc/Gal ratio increased. Also, deletion of atxA, a global regulatory gene, from a pXO2⁻ strain resulted in cell walls with an even greater level of Glc.     

Multilocus sequence typing for phylogenetic view and <Emphasis Type="Italic">vip</Emphasis> gene diversity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains of the Assam soil of North East India

An agriculturally important insecticidal bacterium, Bacillus thuringiensis have been isolated from the soil samples of various part of Assam including the Kaziranga National Park. Previously, the isolates were characterized based on morphology, 16S rDNA sequencing, and the presence of the various classes’ crystal protein gene(s). In the present study, the phylogenetic analysis of a few selected isolates was performed by an unambiguous and quick method called the multiple locus sequence typing (MLST). A known B. thuringiensis strain kurstaki 4D4 have been used as a reference strain for MLST. A total of four the MLST locus of housekeeping genes, recF, sucC, gdpD and yhfL were selected. A total of 14 unique sequence types (STs) was identified. A total number of alleles identified for the locus gdpD and sucC was 12, followed by locus yhfL was 11, however, only 6 alleles were detected for the locus recF. The phylogenetic analysis using MEGA 7.0.26 showed three major lineages. Approximately, 87% of the isolates belonged to the STs corresponding to B. thuringiensis, whereas two isolates, BA07 and BA39, were clustered to B. cereus. The isolates were also screened for the diversity of vegetative insecticidal protein (vip) genes. In all, 8 isolates showed the presence of vip1, followed by 7 isolates having vip2 and 6 isolates for vip3 genes. The expression of Vip3A proteins was analyzed by western blot analyses and expression of the Vip3A protein was observed in the isolate BA20. Thus, the phylogenetic relationship and diversity of Bt isolates from Assam soil was established based on MLST, in addition, found isolates having vip genes, which could be used for crop improvement.     

Diagnostic properties of three conventional selective plating media for selection of <Emphasis Type="Italic">Bacillus cereus</Emphasis>, <Emphasis Type="Italic">B. thuringiensis</Emphasis> and <Emphasis Type="Italic">B. weihenstephanensis</Emphasis>

The aim of this study was to assess the diagnostic properties of the two selective plating media and a chromogenic medium for identification of Bacillus cereus. The 324 isolates were B. cereus (37%), Bacillus weihenstephanensis (45%) or Bacillus thuringiensis (18%), as identified by a new combination of techniques. All isolates were growing on mannitol–egg yolk–polymyxin agar (MYP), and they did not form acid from mannitol. However, a significant lower number of B. thuringiensis isolates did not show lecithinase activity. All isolates were also growing on polymyxin–egg yolk–mannitol–bromothymol blue agar (PEMBA); however, 11% isolates indicated that they did produce acid from mannitol, and 15% isolates did not show any lecithinase activity. Five of the isolates did not grow at all on the chromogenic agar, and 14 of the growing isolates were β-glucosidase negative. It is concluded that the two recommended selective plating media MYP and PEMBA for detection of B. cereus group bacteria both have their limitations for identification of some B. cereus, B. weihenstephanensis or B. thuringiensis. However, MYP is preferable compared to PEMBA. The chromogenic medium has its own advantages and limitations, and some of the limitations seem to be solved by incubation at 30°C instead of the recommended 37°C.     

Prevalence,Genetic Diversity,and Host Range of Tectiviruses among Members of the Bacillus cereus Group

GIL01, Bam35, GIL16, AP50, and Wip1 are tectiviruses preying on the Bacillus cereus group. Despite the significant contributions of phages in different biological processes, little is known about the dealings taking place between tectiviruses and their Gram-positive bacterial hosts. Therefore, this work focuses on characterizing the interactions between tectiviruses and the B. cereus group by assessing their occurrence and genetic diversity and evaluating their host range. To study the occurrence of tectiviruses in the B. cereus group, 2,000 isolates were evaluated using primers designed to be specific to two variable regions detected in previously described elements. PCR and propagation tests revealed that tectivirus-like elements occurred in less than 3% of the isolates. Regardless of this limited distribution, several novel tectiviruses were found, and partial DNA sequencing indicated that a greater diversity exists within the family Tectiviridae. Analyses of the selected variable regions, along with their host range, showed that tectiviruses in the B. cereus group can be clustered mainly into two different groups: the ones infecting B. anthracis and those isolated from other B. cereus group members. In order to address the host range of some novel tectiviruses, 120 strains were tested for sensitivity. The results showed that all the tested tectiviruses produced lysis in at least one B. cereus sensu lato strain. Moreover, no simple relationship between the infection patterns of the tectiviruses and their diversity was found.     

Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.     

Bartonella chomelii Is the Most Frequent Species Infecting Cattle Grazing in Communal Mountain Pastures in Spain

The presence of Bartonella spp. was investigated in domestic ungulates grazing in communal pastures from a mountain area in northern Spain, where 18.3% (17/93) of cattle were found to be positive by PCR combined with a reverse line blot (PCR/RLB), whereas sheep (n = 133) or horses (n = 91) were found not to be infected by this pathogen. Bartonella infection was significantly associated with age, since older animals showed a higher prevalence than heifers and calves. In contrast to other studies, B. chomelii was the most frequent species found in cattle (14/17), while B. bovis was detected in only three animals. Moreover, 18 B. chomelii isolates and one B. bovis isolate were obtained from nine animals. Afterwards, B. chomelii isolates were characterized by a multilocus sequence typing (MLST) method which was adapted in this study. This method presented a high discrimination power, identifying nine different sequence types (STs). This characterization also showed the presence of different STs simultaneously in the same host and that STs had switched over time in one of the animals. In addition, B. chomelii STs seem to group phylogenetically in two different lineages. The only B. bovis isolate was characterized with a previously described MLST method. This isolate corresponded to a new ST which is located in lineage I, where the B. bovis strains infecting Bos taurus subsp. taurus are grouped. Further studies on the dynamics of Bartonella infection in cattle and the potential ectoparasites involved in the transmission of this microorganism should be performed, improving knowledge about the interaction of Bartonella spp. and domestic ungulates.     

Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.     

Bioefficacy of some Rhizobactrial isolates against sorghum root Rot pathogen Bipolaris sorokiniana

This study carried out to identify certain microbial allelochemicals with antifungal activity of some rhziobacterial isolates against Bipolaris sorokiniana fungi. The fungicidal activity of isolated microbe metabolites was compared based on inhibition % of fungal growth. Results showed that ethyl acetate crude extracts with two concentrations (500 and 1000 ppm) of Pseudomonas geniculata (SC) and Bacillus cereus (S4) were the most efficient isolates recorded inhibition % 33.62 and 52.59% followed by S4 (Bacillus cereus (ATCC 14579) which achieved inhibition % 33.62 and 46.55% at the same concentrations, respectively. After 4 days.The constituents analyzed by LC-MS/MS and FTIR of the ethyl acetate extracts of the Pseudomonas geniculata ATCC19374 were afforded aminobutyric acid, 1,4-benzoquinone, coumaric acid, sinapic acid, tryptophan amino acid, Succinic acid and ferulic acid. While, the secondary metabolites of (Bacillus cereus ATCC 14579 extract were aminobutyric acid, 1,4-benzoquinone, coumaric acids, sinapic acid, ferulic acid and benzoic acid. Results indicated that the isolates of Pseudomonas geniculata ATCC19374 and Bacillus cereus ATCC 14579 could be use as a good element in plant root rot pathogen Bipolaris sorokiniana management.     

Multilocus sequence typing reveals that Bacillus cereus strains isolated from clinical infections have distinct phylogenetic origins

Eight strains of Bacillus cereus isolated from bacteremia and soft tissue infections were assigned to seven sequence types (STs) by multilocus sequence typing (MLST). Two strains from different locations had identical STs. The concatenated sequences of the seven STs were aligned with 65 concatenated sequences from reference STs and a neighbor-joining tree was constructed. Two strains were distantly related to all reference STs. Three strains were recovered in a clade that included Bacillus anthracis, B. cereus and rare Bacillus thuringiensis strains while the other three strains were assigned to two STs that were more closely affiliated to most of the B. thuringiensis STs. We conclude that invasive B. cereus strains do not form a single clone or clonal complex of highly virulent strains.     

Identification of Bacillus anthracis Spore Component Antigens Conserved across Diverse Bacillus cereus sensu lato Strains

We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.The anthrax causing bacterium Bacillus anthracis is a member of the Bacillus cereus sensu lato (s.l.)¹ group, a term given to the polyphyletic species consisting of Bacillus thuringiensis, Bacillus cereus, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides (1). Genomics studies of B. cereus s.l. strains have shown a similar chromosomal gene composition within this group (2–7). Many phenotypes that distinguish B. cereus s.l. members, such as crystalline toxin production (8), emesis in humans (9), and anthrax virulence (10), are encoded by genes on large plasmids. Experimental conjugative transfer of plasmids between B. cereus s.l. strains has been demonstrated in vitro, in complex media, and in vector species (11–13). Therefore there is a concern about transfer of virulence genes between genetic backgrounds creating new pathogen lineages. In this regard, there is an emerging evidence of natural dissemination of the pXO1 and pXO2 plasmids that encode the anthrax lethal toxin and capsule, respectively. For example, B. cereus G9241 carries a pXO1 plasmid and lethal toxin genes almost identical to those in B. anthracis (6), and a B. cereus strain, which causes anthrax-like illness in African great apes, apparently contains both pXO1 and pXO2 plasmids (14).The infectious agent of most if not all human B. cereus s.l. diseases is the spore. The spore is a dormant, environmentally resistant structure that persists in nutrient- or water-limiting conditions. Anthrax infection occurs after introduction of the B. anthracis spore into a skin abrasion or via inhalation or ingestion (10). The spore germinates inside host cells, and the resulting vegetative bacteria express toxins and capsules that elicit an immune response (10, 15, 16). Formation of the B. cereus spore involves asymmetric cell division during which a copy of the genome is partitioned into each of the sister cells. The smaller cell (prespore) develops into mature endospore, and the larger cell (mother cell) contributes to the differentiation process but undergoes autolysis following its completion to release the endospore into the surrounding medium. Synthesis of cortex, coat, and exosporium are a function mainly of the mother cell. The cortex and coat layers are in close proximity to one another, whereas the exosporium tends to appear as an irregularly shaped, loosely attached, balloon-like layer (17–20). The coat and the exosporium contribute to the remarkable resistance of spores to extreme physical and chemical stresses including the exposure to extraterrestrial conditions (21, 22). Recent work on the structure, composition, assembly, and function of the spore coat and exosporium of pathogenic organisms like B. anthracis and B. cereus have highlighted the crucial link that exists between the origin of these layers (19, 23). There are differences in the appearance and thickness of the coat layers among the spores of various strains and species. In some B. thuringiensis strains, the inner coat is laminated but consists of a patchwork of striated packets, appearing either stacked or comblike, and the outer coat is granular (24), whereas in B. anthracis and other B. cereus s.l. isolates the coat appears compact (25–27). The coat layers comprise about 30% of the total proteins present in the spore (19, 28). Intraspecies variation in the structure and composition of the spore surface layers may reflect the environmental conditions under which these spores are formed (29–31).Because the spore is crucial to infection and persistence of B. anthracis and its close relatives, we undertook an investigation of its protein profile variability across the B. cereus s.l. group. Our goal in this study was to identify conserved antigenic spore proteins that may be transitioned in the future as candidates for immunodiagnostics, therapeutics, or vaccines. We used two high throughput approaches: genome-based bioinformatics analysis and comparative proteomics analysis of spores of B. cereus s.l. to select conserved targets. Our analysis revealed a list of conserved spore proteins within B. cereus but relatively few cross-reacting antigens. Two of these spore conserved antigens (Crd and MetN) have not been described previously for B. anthracis.     

Campylobacter spp. Recovered from the Upper Oconee River Watershed, Georgia in a 4-Year Study

Waterways should be considered in the migration routes of Campylobacter, and the genus has been isolated from several water sources. Inferences on migration routes can be made from tracking genetic types in populations found in specific habitats and testing how they are linked to other types. Water samples were taken over a 4-year period from waterways in the Upper Oconee River Watershed, Georgia, to recover isolates of thermophilic Campylobacter. The isolates were typed by multilocus sequence typing (MLST) and analyzed to determine the overall diversity of Campylobacter in that environment. Forty-seven independent isolates were recovered from 560 samples (8.4 %). Two (~4 %) isolates were Campylobacter coli, three (~6 %) isolates were putatively identified as Campylobacter lari, and the remaining 42 (~90 %) were Campylobacter jejuni. The C. jejuni and C. coli isolates were typed by the Oxford MLST scheme. Thirty sequence types (STs) were identified including 13 STs that were not found before in the MLST database, including 24 novel alleles. Of the 17 previously described STs, 10 have been isolated from humans, 6 from environmental water, and 6 from wild birds (five types from multiple sources). Seven sites had multiple positive samples, and on two occasions, the same ST was isolated at the same site. The most common type was STST61 with four isolates, and the most common clonal complex was CC179 with nine isolates. CC179 has been commonly associated with environmental water. Although some Campylobacter STs that were found in the Oconee River engage in widespread migration, most are tightly associated with or unique to environmental water sources.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Distribution of Sequence-Based Types of Legionella pneumophila Serogroup 1 Strains Isolated from Cooling Towers,Hot Springs,and Potable Water Systems in China

Legionella pneumophila serogroup 1 causes Legionnaires'' disease. Water systems contaminated with Legionella are the implicated sources of Legionnaires'' disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n = 96), hot springs (n = 42), and potable water systems (n = 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n = 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.