Application of Fourier Transform Infrared Spectroscopy and Chemometrics for Differentiation of Salmonella enterica Serovar Enteritidis Phage Types

Fourier transform infrared (FT-IR) spectroscopy and chemometric techniques were used to discriminate five closely related Salmonella enterica serotype Enteritidis phage types, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a. Intact cells and outer membrane protein (OMP) extracts from bacterial cell membranes were subjected to FT-IR analysis in transmittance mode. Spectra were collected over a wavenumber range from 4,000 to 600 cm⁻¹. Partial least-squares discriminant analysis (PLS-DA) was used to develop calibration models based on preprocessed FT-IR spectra. The analysis based on OMP extracts provided greater separation between the Salmonella Enteritidis PT1-PT1b, PT4b, and PT6-PT6a groups than the intact cell analysis. When these three phage type groups were considered, the method based on OMP extract FT-IR spectra was 100% accurate. Moreover, complementary local models that considered only the PT1-PT1b and PT6-PT6a groups were developed, and the level of discrimination increased. PT1 and PT1b isolates were differentiated successfully with the local model using the entire OMP extract spectrum (98.3% correct predictions), whereas the accuracy of discrimination between PT6 and PT6a isolates was 86.0%. Isolates belonging to different phage types (PT19, PT20, and PT21) were used with the model to test its robustness. For the first time it was demonstrated that FT-IR analysis of OMP extracts can be used for construction of robust models that allow fast and accurate discrimination of different Salmonella Enteritidis phage types.Over the past 10 years there has been an increase in the incidence of gastrointestinal infections caused by Salmonella enterica serovar Enteritidis, which is now one of the leading S. enterica serotypes worldwide (21, 27). Poultry, poultry products, cattle, and dairy products are the predominant sources of Salmonella-contaminated food products that cause human salmonellosis (28). Large-scale infections continue to occur in developed countries (8). Unrestricted international movement of commercially prepared food and food ingredients and dissimilarities in government and industry food safety controls during the processing, distribution, and marketing of products have surely contributed to the increase in food-borne outbreaks. Salmonella is a tremendous challenge for the agricultural and food processing industries because of its ability to survive under adverse conditions, such as low levels of nutrients and suboptimal temperatures (4, 13).Salmonella Enteritidis isolates can be categorized for epidemiological purposes by using a variety of typing tools (13). These tools include typing techniques such as serological and phage typing (29) and antibiotic resistance patterns (25). These methods are now supplemented by molecular genetics techniques, such as DNA fingerprinting (23), plasmid profiling (16), and pulsed-field gel electrophoresis (26). Phage typing has been used to diagnose Salmonella outbreaks, including S. enterica serovar Typhi and S. enterica serovar Typhimurium outbreaks (29). It is useful to evaluate whether isolates obtained from different sources at different times are similar or distinct in terms of their reactions with a specific collection of bacteriophages used for typing. The correlation between phage type and the source of an epidemic is high (22). Although very effective, existing classification methods are time-consuming, laborious, and expensive, and they often require special training of personnel and expertise, which can prevent a rapid response to the presence of pathogenic bacterial species.Fourier transform infrared (FT-IR) spectroscopy has been successfully used for differentiation and classification of microorganisms at the species and subspecies levels (7, 9, 12, 15, 18, 19, 20). This technique has been shown to have high discriminatory power and allows identification of bacteria at distinct taxonomic levels based on differences in the infrared absorption patterns of microbial cells. FT-IR spectroscopy has been used to differentiate and characterize intact microbial cells based on outer membrane cell components, including lipopolysaccharides (LPS), lipoproteins, and phospholipids (24). Several studies in which S. enterica serotypes have been discriminated using multivariate data analysis and FT-IR spectroscopy have been performed (1, 2, 10, 11). Kim et al. (11) compared the FT-IR spectra of intact cells and the FT-IR spectra of outer membrane protein (OMP) extracts from S. enterica serotypes to discriminate serotypes. Analysis of spectra of OMP extracts in the 1,800- to 1,500-cm⁻¹ region resulted in 100% correct classification of the serotypes investigated.Previously, there have been no reports of differentiation of Salmonella Enteritidis phage types by FT-IR spectroscopy and chemometric methods. To discriminate closely related phage types of Salmonella Enteritidis in this study, intact cells and OMP extracts of bacterial cell membranes were subjected to FT-IR analysis. The isolates analyzed included isolates belonging to five of the phage types of Salmonella Enteritidis found most frequently in Portuguese hospitals in the period from 2004 to 2006, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a (5, 14). Chemometric models were used to discriminate between phage types based on infrared spectra.     

Spontaneous Excision of the Salmonella enterica Serovar Enteritidis-Specific Defective Prophage-Like Element φSE14

Salmonella enterica serovar Enteritidis has emerged as a major health problem worldwide in the last few decades. DNA loci unique to S. Enteritidis can provide markers for detection of this pathogen and may reveal pathogenic mechanisms restricted to this serovar. An in silico comparison of 16 Salmonella genomic sequences revealed the presence of an ∼12.5-kb genomic island (GEI) specific to the sequenced S. Enteritidis strain NCTC13349. The GEI is inserted at the 5′ end of gene ydaO (SEN1377), is flanked by 308-bp imperfect direct repeats (attL and attR), and includes 21 open reading frames (SEN1378 to SEN1398), encoding primarily phage-related proteins. Accordingly, this GEI has been annotated as the defective prophage SE14 in the genome of strain NCTC13349. The genetic structure and location of φSE14 are conserved in 99 of 103 wild-type strains of S. Enteritidis studied here, including reference strains NCTC13349 and LK5. Notably, an extrachromosomal circular form of φSE14 was detected in every strain carrying this island. The presence of attP sites in the circular forms detected in NCTC13349 and LK5 was confirmed. In addition, we observed spontaneous loss of a tetRA-tagged version of φSE14, leaving an empty attB site in the genome of strain NCTC13349. Collectively, these results demonstrate that φSE14 is an unstable genetic element that undergoes spontaneous excision under standard growth conditions. An internal fragment of φSE14 designated Sdf I has been used as a serovar-specific genetic marker in PCR-based detection systems and as a tool to determine S. Enteritidis levels in experimental infections. The instability of this region may require a reassessment of its suitability for such applications.The genus Salmonella comprises a heterogeneous group of Gram-negative bacteria, differentiable by biochemical and serological properties. More than 2,500 Salmonella serovars have been identified according to the serospecificities of the somatic and flagellar antigens. Some serovars, exemplified by Salmonella enterica serovar Typhimurium and S. Enteritidis, can infect a broad range of hosts. However, a subset of serovars, such as S. Typhi, a human-specific pathogen, show a high degree of adaptation to a specific host.In the last few decades, S. Enteritidis has emerged as a major health problem worldwide (31). This pathogen colonizes the reproductive organs of infected birds without causing discernible illness and survives host defenses during the formation of the egg (25, 27). The production of a capsule-like O antigen structure by certain wild-type strains of S. Enteritidis (30, 46) has been associated with reproductive tract tropism and improved survival within eggs (26, 27, 45). Egg contamination can originate before oviposition by direct contamination of the yolk, albumen, or eggshell membranes with bacteria from the infected reproductive organs of the birds or after or during oviposition by penetration of bacteria from contaminated feces through the eggshell (8, 14, 25). Transmission of the bacterium to humans occurs mainly through the consumption of contaminated eggs or egg products (8, 14, 25). Upon infection of a human host, S. Enteritidis causes self-limiting gastroenteritis similar to that caused by other nontyphoidal Salmonella serovars.According to information gathered from 84 countries responding to a global survey conducted by the World Health Organization (WHO), S. Enteritidis and S. Typhimurium accounted for ∼70% of all human and nonhuman isolates of Salmonella reported worldwide between 1995 and 2008. In fact, S. Enteritidis alone accounted for 61.4% of the ∼1.5 million human isolates of Salmonella reported during this period, according to the WHO Global Foodborne Infections Network Country Databank (http://www.who.int/salmsurv). Remarkably, S. Enteritidis is the second most prevalent cause of Salmonella infection in humans, after S. Typhimurium, in the United States (10).The high global prevalence of S. Enteritidis makes the development of a rapid, sensitive, and highly specific detection system critical to collect accurate epidemiologic data. The identification of loci that serve as specific markers for DNA-based identification of this pathogen may also provide insights into pathogenic mechanisms restricted to this serovar. Genomic regions that are unique to given serovars are especially suitable for such epidemiologic detection (3). For instance, Agron and colleagues identified an S. Enteritidis-specific genomic region of ∼4,060 bp adjacent to the ydaO gene, carrying six open reading frames (ORFs) that they designated lygA to lygF (1). A PCR-based assay successfully detected the presence of an internal fragment of this serovar-specific region in most strains in a diverse collection of clinical and environmental S. Enteritidis isolates and not in 73 non-Enteritidis isolates of S. enterica representing 34 different serovars (1). Since then, this region has been widely used as an S. Enteritidis-specific molecular marker in the development of several PCR-based assays for detection and epidemiological typing of Salmonella serovars in clinical and environmental samples (2, 11, 32, 37, 44, 53). Recently, an S. Enteritidis-specific real-time quantitative PCR (qPCR) assay based on the detection of this region was developed (15). This qPCR assay has been used in a series of studies of the distribution and replication kinetics of S. Enteritidis in experimentally infected animals (16-21).We performed a bioinformatic study to identify genomic regions specific to S. Enteritidis and found a genomic island (GEI) that includes the S. Enteritidis-specific locus lyg (1). This island has been annotated as the defective prophage SE14 in the genome of S. Enteritidis strain NCTC13349 (52). Although we demonstrate that the location in the genome and the overall genetic structure of the island are conserved in wild-type isolates of S. Enteritidis from different origins, we detected strains that do not carry the island in their genomes. Finally, we demonstrate here that the island corresponds to an unstable element that undergoes spontaneous excision from the genome of S. Enteritidis under standard growth conditions.     

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).     

Inhibition of the Early Stage of Salmonella enterica Serovar Enteritidis Biofilm Development on Stainless Steel by Cell-Free Supernatant of a Hafnia alvei Culture

Compounds present in Hafnia alvei cell-free culture supernatant cumulatively negatively influence the early stage of biofilm development by Salmonella enterica serovar Enteritidis on stainless steel while they also reduce the overall metabolic activity of S. Enteritidis planktonic cells. Although acylhomoserine lactones (AHLs) were detected among these compounds, the use of several synthetic AHLs was not able to affect the initial stage of biofilm formation by this pathogen.Biofilms are groups of bacteria encased in a self-produced extracellular matrix (5, 6). Biofilms formed on stainless steel (SS) surfaces in food-processing areas are of great importance since they may lead to food spoilage and transmission of diseases (2, 16). This sessile mode of life allows bacteria to enjoy a number of advantages, such as increased resistance to antimicrobial agents (9, 12). Notably, it is widely accepted that bacteria (both planktonic and biofilm cells) communicate by releasing and sensing signaling compounds in a process commonly known as quorum sensing (13, 18, 24).Salmonella enterica serovar Enteritidis is one of the most important bacterial pathogens worldwide (7, 17). Hafnia alvei are frequent psychrotrophic members of the Enterobacteriaceae community in meat products, playing a role in their spoilage, while they have been shown to be capable of producing signaling compounds (3). In this study, in order to determine any possible influence of compounds produced by H. alvei on the biofilm-forming ability of S. Enteritidis, the latter was left to develop biofilm on SS surfaces in the presence of conditioned medium obtained after the growth of the former. Biofilm formation was assessed directly by detaching cells and enumerating them and, also, indirectly by automated conductance measurements.     

Evidence for Mucin-Like Glycoproteins That Tether Sporozoites of Cryptosporidium parvum to the Inner Surface of the Oocyst Wall

Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls.Cryptosporidium parvum and the related species Cryptosporidium hominis are apicomplexan parasites, which are spread by the fecal-oral route in contaminated water and cause diarrhea, particularly in immunocompromised hosts (1, 12, 39, 47). The infectious and diagnostic form of C. parvum is the oocyst, which has a single, multilayered, spherical wall that surrounds four sporozoites, the invasive forms (14, 27, 31). The outermost layer of the C. parvum oocyst wall is most often absent from electron micrographs, as it is labile to bleach used to remove contaminating bacteria from C. parvum oocysts (27). We will refer to this layer as the outer veil, which is the term used for a structure with an identical appearance on the surface of the oocyst wall of another apicomplexan parasite, Toxoplasma gondii (10). At the center of the C. parvum oocyst wall is a protease-resistant and rigid bilayer that contains GalNAc (5, 23, 43). When excysting sporozoites break through the oocyst wall, the broken edges of this bilayer curl in, while the overall shape of the oocyst wall remains spherical.The inner, moderately electron-dense layer of the C. parvum oocyst wall is where the Cryptosporidium oocyst wall proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) have been localized with monoclonal antibodies (4, 20, 28, 32). COWPs, which have homologues in Toxoplasma, are a family of nine proteins that contain polymorphic Cys-rich and His-rich repeats (37, 46). Finally, on the inner surface of C. parvum oocyst walls are knob-like structures, which cross-react with an anti-oocyst monoclonal antibody (11).Like other apicomplexa (e.g., Toxoplasma and Plasmodium), sporozoites of C. parvum are slender, move by gliding motility, and release adhesins from apical organelles when they invade host epithelial cells (1, 8, 12, 39). Unlike other apicomplexa, C. parvum parasites are missing a chloroplast-derived organelle called the apicoplast (1, 47, 49). C. parvum sporozoites have on their surface unique mucin-like glycoproteins, which contain Ser- and Thr-rich repeats that are polymorphic and may be modified by O-linked GalNAc (4-7, 21, 25, 26, 30, 32, 34, 35, 43, 45). These C. parvum mucins, which are highly immunogenic and are potentially important vaccine candidates, include gp900 and gp40/gp15 (4, 6, 7, 25, 26). gp40/gp15 is cleaved by furin-like proteases into two peptides (gp40 and gp15), each of which is antigenic (42). gp900, gp40, and gp15 are shed from the surface of the C. parvum sporozoites during gliding motility (4, 7, 35).The studies presented here began with electron microscopic observations of C. parvum oocysts stained with ruthenium red (23), which revealed novel fibrils or tethers that extend radially from the inner surface of the oocyst wall to the outer surface of sporozoites. We hypothesized that at least some of these fibrillar tethers might be the antigenic mucins, which are abundant on the surface of C. parvum sporozoites. To test this hypothesis, we used mass spectroscopy to identify oocyst wall proteins and sporozoite glycoproteins and used deconvolving and immunoelectron microscopy (immuno-EM) with lectins and anti-C. parvum antibodies to directly label the tethers.     

Comparison and Utilization of Repetitive-Element PCR Techniques for Typing Lactobacillus Isolates from the Chicken Gastrointestinal Tract

Three repetitive-element PCR techniques were evaluated for the ability to type strains of Lactobacillus species commonly identified in the chicken gastrointestinal tract. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) produced species- and strain-specific profiles for Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri isolates. The technique typed strains within these species equally as well as pulsed-field gel electrophoresis. DNA concentration and quality did not affect the ERIC-PCR profiles, indicating that this method, unlike other high-resolution methods, can be adapted to high-throughput analysis of isolates. Subsequently, ERIC-PCR was used to type Lactobacillus species diversity of a large collection of isolates derived from chickens grown under commercial and necrotic enteritis disease induction conditions. This study has illustrated, for the first time, that there is great strain diversity within each Lactobacillus species present and has revealed that chickens raised under commercial conditions harbor greater species and strain diversity than chickens raised under necrotic enteritis disease induction conditions.Lactobacilli are normal inhabitants within the microflora of the chicken gastrointestinal tract (GIT) (27, 39). Species frequently identified within the chicken GIT include Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri (1, 7, 27). The first three of these species are members of the Lactobacillus acidophilus complex (LAC) (22, 32), a closely related group of species which are difficult to differentiate using traditional techniques, such as physiological and biochemical tests (34). While molecular methods, such as DNA-DNA hybridization (22), ribotyping (71), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (19), randomly amplified polymorphic DNA PCR (19), and 16S rRNA gene sequencing (41), have been used with some success, many of these techniques are not readily adaptable to high-throughput applications required for large-scale ecological studies.While numerous studies have reported Lactobacillus species distribution within the chicken GIT, the strain diversity within species has not been explored. Previously, Hagen et al. (28) investigated L. gallinarum isolates present within the crops of commercial chickens, revealing a high level of strain diversity among the isolates examined (17 strains represented among 38 isolates). These results indicate that there could be great diversity within and among the lactobacilli present within the chicken GIT. Examining and typing large numbers of lactobacilli from the chicken GIT to the strain level may facilitate a better understanding of microflora dynamics and niche competition. These studies may also result in the identification of strains which could be used in competitive exclusion applications and potentially in the development of probiotics or live vectors for the delivery of therapeutic recombinant proteins to specific sites within the chicken GIT.Lactobacilli have been proposed as possible competitive exclusion agents or probiotics (30, 36, 42) against Clostridium perfringens, the causative agent of necrotic enteritis (NE) in broiler chickens. The withdrawal of antimicrobial growth promoters in Europe has led to an increase in the incidence of NE (10, 64), prompting the investigation of alternative methods for controlling C. perfringens in the broiler chicken GIT. Various models have been developed to study NE under research conditions, as recently reviewed by Dahiya et al. (15). The conditions used in these NE models and the effects they have on GIT microflora may adversely impact the identification of antimicrobial alternatives, such as probiotic strains, for use within commercial broiler chickens. Application of a high-throughput typing method capable of distinguishing species and strains is needed to determine if differences exist between the Lactobacillus populations of chickens raised under NE and commercial conditions.While pulsed-field gel electrophoresis (PFGE) has been used widely for genotyping Lactobacillus strains (49, 50, 65), it is time-consuming, labor-intensive, expensive, and suitable only for low-throughput analysis of isolates (54, 65). In contrast, repetitive-element PCR (Rep-PCR) has been developed for genotypically fingerprinting bacteria and is a fast and reliable high-throughput genotyping system (66, 67). Rep-PCR has been used successfully to identify strains of a variety of genera (33, 44, 48, 55). Several Rep-PCR primers, including the repetitive extragenic palindromic (REP) primers (6, 9, 16, 65), the enterobacterial repetitive intergenic consensus (ERIC) primers (6, 65), and the (GTG)₅ primer (24, 40, 62), have been used in the typing of lactobacilli in studies which have generally focused on species important to the dairy industry and food fermentations. Very few studies have examined the application of Rep-PCR to species commonly identified in the chicken GIT (62, 65). To our knowledge, only a single study has actually applied Rep-PCR to type Lactobacillus isolates from chickens (62) for the identification of potential probiotics to control Salmonella enterica serovar Enteritidis in egg-laying hens.The aim of this study was to investigate whether Rep-PCR could be used to analyze Lactobacillus species and strains in the chicken GIT. Several Rep-PCR techniques [REP-, ERIC-, and (GTG)₅-PCR] were compared for the ability to type strains of several Lactobacillus species commonly isolated from the chicken GIT (L. crispatus, L. gallinarum, L. johnsonii, and L. reuteri). ERIC-PCR was able to simultaneously type isolates to the species and strain levels, and its strain differentiation ability was comparable with that of PFGE. ERIC-PCR was further applied to high-throughput analysis of a large number of isolates collected from chickens raised under NE and commercial conditions.     

Selection of a Bacillus pumilus Strain Highly Active against Ceratitis capitata (Wiedemann) Larvae

Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), the Mediterranean fruit fly (medfly), is one of the most important fruit pests worldwide. The medfly is a polyphagous species that causes losses in many crops, which leads to huge economic losses. Entomopathogenic bacteria belonging to the genus Bacillus have been proven to be safe, environmentally friendly, and cost-effective tools to control pest populations. As no control method for C. capitata based on these bacteria has been developed, isolation of novel strains is needed. Here, we report the isolation of 115 bacterial strains and the results of toxicity screening with adults and larvae of C. capitata. As a result of this analysis, we obtained a novel Bacillus pumilus strain, strain 15.1, that is highly toxic to C. capitata larvae. The toxicity of this strain for C. capitata was related to the sporulation process and was observed only when cultures were incubated at low temperatures before they were used in a bioassay. The mortality rate for C. capitata larvae ranged from 68 to 94% depending on the conditions under which the culture was kept before the bioassay. Toxicity was proven to be a special characteristic of the newly isolated strain, since other B. pumilus strains did not have a toxic effect on C. capitata larvae. The results of the present study suggest that B. pumilus 15.1 could be considered a strong candidate for developing strategies for biological control of C. capitata.The Mediterranean fruit fly (medfly), Ceratitis capitata, is considered a highly invasive agricultural and economically important pest throughout the world. In less than 200 years the range of this species has expanded from its native habitat in sub-Saharan Africa, and it has become a cosmopolitan species (26) that is present on five continents (14, 46). The wide distribution of the medfly is attributed, among other things, to its remarkably polyphagous behavior (more than 300 host plants have been reported) (43), to its resistance to cold climates (65), and to successful establishment after multiple introductions (30, 49) as a result of the increasing frequency of global trade (46).Medfly infestations cause serious economic losses and sometimes result in complete loss of crops (76). Numerous methods have been tried to control medfly populations, including chemical products, such as malathion and other organophosphate insecticides (4, 8), classic biological control programs based on the release of some of parasitoids and predators (38, 41, 44), toxic baits (2, 13, 31, 32, 35, 56), mass trapping systems (24, 51), the sterile insect technique (7, 34, 61, 63, 72, 73), and development of integrated strategies of management (71). In spite of all of these attempts, control of Mediterranean fruit fly populations has been ineffective, and losses associated with this pest worldwide are constantly increasing (21, 46).Insecticides based on microbial agents (bacteria, fungi, and viruses) are a promising alternative that has received a great deal of attention for control of C. capitata (5, 13, 18, 40, 55), but so far no such insecticide has reached a commercial stage. Among the microbial insecticides, bacteria are very successful agents in biological control programs (17, 29). The entomopathogenic bacteria belonging to the genus Bacillus are natural agents used for biological control of invertebrate pests and are the basis of many commercial insecticides. Three species of the genus Bacillus have been mass produced and commercialized: Bacillus sphaericus, Bacillus thuringiensis, and Paenibacillus popilliae (formerly Bacillus popilliae) (29, 54). These organisms have different spectra and levels of activity that are correlated with the nature of the toxins, which are very frequently produced during sporulation (16, 17). B. thuringiensis was the first Bacillus species used in biological control programs for pests and human vector disease insects (17, 62). During its stationary phase, this Gram-positive, aerobic, ubiquitous, endospore-forming bacterium produces parasporal crystalline inclusions composed mainly of two types of insecticidal proteins (Cry and Cyt toxins) (62) that are toxic to a variety of insects, in some cases at the species level.There have been some reports of B. thuringiensis strains active against other fruit flies (3, 37, 58, 59, 67), but there has been no report of any Bacillus strain with activity against C. capitata.The aim of this study was to search for novel bacteria belonging to the genus Bacillus, specifically B. thuringiensis, with activity against adults and larvae of C. capitata that could be used as biological control agents. Isolation of 115 bacterial strains, evaluation of the insecticidal activities of these strains, and identification of a novel strain of Bacillus pumilus that is highly toxic to C. capitata larvae are reported here. In addition, we found that toxicity was observed only when cultures of B. pumilus strain 15.1 were exposed to low temperatures. The isolation of this novel pathogenic strain could be important for future development of biotechnological strategies aimed at reducing the economic losses caused by C. capitata.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

African 1, an Epidemiologically Important Clonal Complex of Mycobacterium bovis Dominant in Mali,Nigeria, Cameroon,and Chad

We have identified a clonal complex of Mycobacterium bovis present at high frequency in cattle in population samples from several sub-Saharan west-central African countries. This closely related group of bacteria is defined by a specific chromosomal deletion (RDAf1) and can be identified by the absence of spacer 30 in the standard spoligotype typing scheme. We have named this group of strains the African 1 (Af1) clonal complex and have defined the spoligotype signature of this clonal complex as being the same as the M. bovis BCG vaccine strain but with the deletion of spacer 30. Strains of the Af1 clonal complex were found at high frequency in population samples of M. bovis from cattle in Mali, Cameroon, Nigeria, and Chad, and using a combination of variable-number tandem repeat typing and spoligotyping, we show that the population of M. bovis in each of these countries is distinct, suggesting that the recent mixing of strains between countries is not common in this area of Africa. Strains with the Af1-specific deletion (RDAf1) were not identified in M. bovis isolates from Algeria, Burundi, Ethiopia, Madagascar, Mozambique, South Africa, Tanzania, and Uganda. Furthermore, the spoligotype signature of the Af1 clonal complex has not been identified in population samples of bovine tuberculosis from Europe, Iran, and South America. These observations suggest that the Af1 clonal complex is geographically localized, albeit to several African countries, and we suggest that the dominance of the clonal complex in this region is the result of an original introduction into cows naïve to bovine tuberculosis.Mycobacterium bovis causes bovine tuberculosis (TB), an important disease of domesticated cattle that has a major economic and health impact throughout the world (61, 64, 65). The pathogen is a member of the Mycobacterium tuberculosis complex, which includes many species and subspecies that cause similar pathologies in a variety of mammalian hosts. The most notable member of the complex is M. tuberculosis, the most important bacterial pathogen of humans. In contrast to M. tuberculosis, which is largely host restricted to humans, M. bovis is primarily maintained in bovids, in particular, domesticated cattle, although the pathogen can frequently be recovered from other mammals, including humans (61). Bovine TB is found in cattle throughout the world and has been reported on every continent where cattle are farmed (3).Bovine TB has been reduced or eliminated from domestic cattle in many developed countries by the application of a test-and-cull policy that removes infected cattle (3, 8, 16, 17, 61, 64, 65). However, in Africa, although bovine TB is known to be common in both cattle and wildlife, control policies have not been enforced in many countries due to cost implications, lack of capacity, and infrastructure limitations (8, 16, 17, 57). In 1998, Cosivi et al. reported of bovine TB, “Of all nations in Africa, only seven apply disease control measures as part of a test-and-slaughter policy and consider bovine TB a notifiable disease; the remaining 48 control the disease inadequately or not at all” (16). In the intervening years, the situation is not thought to have improved (8); however, preliminary surveys of bovine TB have been carried out in some African countries (4, 7, 12, 37, 44, 49, 53, 54, 56).The most common epidemiological molecular-typing method applied to strains of M. bovis is spoligotyping. This method identifies polymorphism in the presence of spacer units in the direct-repeat (DR) region in strains of the M. tuberculosis complex (36, 67). The DR is composed of multiple, virtually identical 36-bp regions interspersed with unique DNA spacer sequences of similar size (direct variant repeat [DVR] units). Spacer sequences are unique to the DR region, and copies are not located elsewhere in the chromosome (68). The DR region may contain over 60 DVR units; however, 43 of the spacer units were selected from the spacer sequences of the M. tuberculosis reference strain H37Rv and M. bovis BCG strain P3 and are used in the standard application of spoligotyping to strains of the M. tuberculosis complex (29, 36). The DR region is polymorphic because of the loss (deletion) of single or multiple spacers, and each spoligotype pattern from strains of M. bovis is given an identifier (http://www.Mbovis.org).Several studies of the DR regions in closely related strains of M. tuberculosis have concluded that the evolutionary trend for this region is primarily loss of single DVRs or multiple contiguous DVRs (22, 29, 68); duplication of DVR units or point mutations in spacer sequences were found to be rare. The loss of discrete units observed by Groenen et al. (29) led them to suggest that the mechanism for spacer loss was homologous recombination between repeat units. However, a study by Warren et al. (69) suggested that for strains of M. tuberculosis, insertion of IS6110 sequences into the DR region and recombination between adjacent IS6110 elements were more important mechanisms for the loss of spacer units.The population structure of the M. tuberculosis group of organisms is apparently highly clonal, without any transfer and recombination of chromosomal sequences between strains (15, 30, 60, 61). In a strictly clonal population, the loss by deletion of unique chromosomal DNA cannot be replaced by recombination from another strain, and the deleted region will act as a molecular marker for the strain and all its descendants. Deletions of specific chromosomal regions (regions of difference [RDs] or large sequence polymorphisms) have been very successful at identifying phylogenetic relationships in the M. tuberculosis complex (11, 25, 26, 35, 48, 50, 61, 62, 66). However, because the loss of spoligotype spacer sequences is so frequent, identical spoligotype patterns can occur independently in unrelated lineages (homoplasy), and therefore, the deletion of spoligotype spacers may be an unreliable indicator of phylogenetic relationship (61, 69).In samples of M. bovis strains from Cameroon, Nigeria, Chad, and Mali, spoligotyping was used to show that many of the strains had similar spoligotype patterns that lacked spacer 30, and it has been suggested that strains from these four countries are phylogenetically related (12, 18, 49, 53). We have extended the previous observations of spoligotype similarities between strains from these countries and confirmed the existence of a unique clonal complex of M. bovis, all descended from a single strain in which a specific deletion of chromosomal DNA occurred. We have named this clonal complex of M. bovis strains African 1 (Af1), and we show that this clonal complex is dominant in these four west-central African countries but rare in eastern and southern Africa. Extended genotyping, using variable-number tandem repeats (VNTR), of strains with the most common spoligotype patterns suggests that each of these four west-central African countries has a unique population structure. Evolutionary scenarios that may have led to the present day distribution of the Af1 clonal complex are discussed.