Identification of Lactobacillus strains with probiotic features from the bottlenose dolphin (Tursiops truncatus).

AIMS: In order to develop complementary health management strategies for marine mammals, we used culture-based and culture-independent approaches to identify gastrointestinal lactobacilli of the common bottlenose dolphin, Tursiops truncatus. METHODS AND RESULTS: We screened 307 bacterial isolates from oral and rectal swabs, milk and gastric fluid, collected from 38 dolphins in the U.S. Navy Marine Mammal Program, for potentially beneficial features. We focused our search on lactobacilli and evaluated their ability to modulate TNF secretion by host cells and inhibit growth of pathogens. We recovered Lactobacillus salivarius strains which secreted factors that stimulated TNF production by human monocytoid cells. These Lact. salivarius isolates inhibited growth of selected marine mammal and human bacterial pathogens. In addition, we identified a novel Lactobacillus species by culture and direct sequencing with 96·3% 16S rDNA sequence similarity to Lactobacillus ceti. CONCLUSIONS: Dolphin-derived Lact. salivarius isolates possess features making them candidate probiotics for clinical studies in marine mammals. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to isolate lactobacilli from dolphins, including a novel Lactobacillus species and a new strain of Lact. salivarius, with potential for veterinary probiotic applications. The isolation and identification of novel Lactobacillus spp. and other indigenous microbes from bottlenose dolphins will enable the study of the biology of symbiotic members of the dolphin microbiota and facilitate the understanding of the microbiomes of these unique animals.

Host Genotype Links to Salivary and Gut Microbiota by Periodontal Status.

Limited evidence describing how host genetic variants affect the composition of the microbiota is currently available. The aim of this study was to assess the associations between a set of candidate host genetic variants and microbial composition in both saliva and gut in the TwinsUK registry. A total of 1,746 participants were included in this study and provided stool samples. A subset of 1,018 participants also provided self-reported periodontal data, and 396 of those participants provided a saliva sample. Host DNA was extracted from whole-blood samples and processed for Infinium Global screening array, focusing on 37 selected single-nucleotide polymorphisms (SNPs) previously associated with periodontitis. The gut and salivary microbiota of participants were profiled using 16S ribosomal RNA amplicon sequencing. Associations between genotype on the selected SNPs and microbial outcomes, including α diversity, ß diversity, and amplicon sequence variants (ASVs), were investigated in a multivariate mixed model. Self-reported periodontal status was also compared with microbial outcomes. Downstream analyses in gut microbiota and salivary microbiota were carried out separately. IL10 rs6667202 and VDR 2228570 SNPs were associated with salivary α diversity, and SNPs in IL10, HSA21, UHRF2, and Fc-γR genes were associated with dissimilarity matrix generated from salivary ß diversity. The SNP that was associated with the greatest number of salivary ASVs was VDR 2228570 followed by IL10 rs6667202, and that of gut ASVs was NPY rs2521364. There were 77 salivary ASVs and 39 gut ASVs differentially abundant in self-reported periodontal disease versus periodontal health. The dissimilarity between saliva and gut microbiota within individuals appeared significantly greater in self-reported periodontal cases compared to periodontal health. IL10 and VDR gene variants may affect salivary microbiota composition. Periodontal status may drive variations in the salivary microbiota and possibly, to a lesser extent, in the gut microbiota.

Bordetella pertussis filamentous hemagglutinin interacts with a leukocyte signal transduction complex and stimulates bacterial adherence to monocyte CR3 (CD11b/CD18).

Bordetella pertussis, the causative agent of whooping cough, adheres to human monocytes/macrophages by means of a bacterial surface-associated protein, filamentous hemagglutinin (FHA) and the leukocyte integrin, complement receptor 3 (CR3, alpha M beta 2, CD11b/CD18). We show that an FHA Arg-Gly-Asp site induces enhanced B. pertussis binding to monocytes, and that this enhancement is blocked by antibodies directed against CR3. Enhancement requires a monocyte signal transduction complex, composed of leukocyte response integrin (alpha? beta 3) and integrin-associated protein (CD47). This complex is known to upregulate CR3 binding activity. Thus, a bacterial pathogen enhances its own attachment to host cells by coopting a host cell signaling pathway.

The search for unrecognized pathogens.

The distribution and diversity of microorganisms in the world are far greater than have been previously appreciated. Molecular, cultivation-independent methods have played a key role in this insight. To what extent do humans remain ignorant of microbial diversity within the human body and the settings in which microorganisms cause human disease? In addition to implicating microbial agents in nontraditional infectious diseases, the use of methods such as broad-range polymerase chain reaction, representational difference analysis, expression library screening, and host gene expression profiling may force a reassessment of the concepts of microbial disease causation.

How the host 'sees' pathogens: global gene expression responses to infection.

Innate immune responses to pathogens are believed to be patterned and stereotyped. Adaptive responses display variety but in relatively few types of products and with limited numbers of mechanisms. Is this apparent disparity between microbial pathogen diversity and a restricted set of host responses an accurate picture of infection or is it the result of a limited collection of analytic tools? DNA microarray technology permits one to address simple descriptive questions about global gene expression inside cells. In particular, it offers an opportunity to examine the relationship between host and pathogen in much greater detail than has been possible previously. One can now ask, firstly, how a host cell or organism 'sees' a microbial pathogen from the viewpoint of gene expression responses and, secondly, at what level it is able to discriminate between different agents. Other potential insights to be reaped include the identification of microbial determinants of the host response, the temporal features of the 'conversation' between host and pathogen, novel strategies for therapeutic and prophylactic intervention and prognostic markers of outcome.

Comparing functional genomic datasets: lessons from DNA microarray analyses of host-pathogen interactions.

Functional genomic technologies such as high density DNA microarrays allow biologists to study the structure and behavior of thousands of genes in a single experiment. One of the fields in which microarrays have had an increasingly important impact is host-pathogen interactions. Early investigations in this area over the past two years not only emphasize the utility of this approach, but also highlight the stereotyped gene expression responses of different host cells to diverse infectious stimuli, and the potential value of broad dataset comparisons in revealing fundamental features of innate immunity. The comparative analysis of recently published datasets involving human gene expression responses to two bacterial respiratory pathogens illustrates many of these points. Comparisons between these large, highly parallel sets of experimental observations also emphasize important technical and experimental design issues as future challenges.

The meaning and impact of the human genome sequence for microbiology.

The characterization of life is immeasurably enhanced by determination of complete genome sequences. For organisms that engage in intimate interactions with others, the genome sequence from one participant, and associated tools, provide unique insight into its partner. We discuss how the human genome sequence will further our understanding of microbial pathogens and commensals, and vice versa. We also propose criteria for implicating a host gene in microbial pathogenesis, and urge consideration of a'second human genome project'.

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism.

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.

Cyclospora.

Although Cyclospora infection has been documented in humans worldwide since at least 1977, it is only in the past 2 years that this organism has come into prominence as a result of major foodborne outbreaks in the United States and Canada. Cyclospora causes significant gastrointestinal disease in immunocompetent and immunocompromised hosts and can be successfully treated with trimethoprim-sulfamethoxazole. The infection is under-recognized because our methods for diagnosis are rudimentary and insensitive. The mechanisms by which the parasite causes disease, the range of animal hosts, and the natural reservoir are unknown. Cyclospora is a unique coccidian parasite that has just begun to emerge; as yet, we have no clue as to where it comes from or where it hides.

Localization of Mycobacterium avium-intracellulare within a skin lesion of bacillary angiomatosis in a patient with AIDS.

We report a 39-year-old man who had AIDS and who presented with an unusual cutaneous vascular lesion, which was clinically thought to be Kaposi's sarcoma. Histologically, the lesion was characterized by capillary proliferation and a mixed inflammatory infiltrate that included numerous histiocytes. The lesion was found to contain slender intracellular acid-fast bacilli, as well as plump extracellular Warthin-Starry-positive bacilli. The acid-fast bacilli were confirmed to be Mycobacterium avium-intracellulare by subsequent positive blood cultures for this organism. To further investigate the lesion, polymerase chain reaction DNA amplification and sequencing was performed, and the lesion was found to contain DNA sequences identical to those previously established for the agent of bacillary angiomatosis. The lesion is thought to represent a lesion of bacillary angiomatosis with secondary involvement by M. avium-intracellulare.

Emerging infections and newly-recognised pathogens.

Clinicians and microbiologists have for many years relied on growth and characterisation of micro-organisms in the laboratory as the major method for their detection and identification, but reliance upon microbial growth in the laboratory has probably significantly limited our ability to recognise important pathogenic micro-organisms. The traditional methods are often slow, non-specific and insensitive, and sometimes discriminate poorly among microbial species and strains. It is now known that the evolutionary ancestry and interrelationships of all living organisms can be reliably inferred from sequences in their genetic material. Highly conserved sequences characterise broad phylogenetic groups and variable sequences allow specific identification. Sequence-based methods combined with DNA amplification methods, such as the polymerase chain reaction (PCR), have led to powerful molecular identification techniques such as consensus nucleic acid amplification and representational difference analysis. These methods allow one to detect and isolate informative gene sequences from occult microbial pathogens in human tissues. Sequence-based methods are often quicker, more sensitive and more specific than traditional methods not only in detecting known microbial pathogens, but also in identifying previously-uncharacterised micro-organisms. Widespread, organised use of these methods will reveal new emerging microbial pathogens, implicate microbes in the aetiology of poorly-understood chronic inflammatory diseases and significantly expand our understanding of microbial diversity.

Species- and strain-specific control of a complex, flexible regulon by Bordetella BvgAS.

The Bordetella master virulence regulatory system, BvgAS, controls a spectrum of gene expression states, including the virulent Bvg(+) phase, the avirulent Bvg(-) phase, and at least one Bvg-intermediate (Bvg(i)) phase. We set out to define the species- and strain-specific features of this regulon based on global gene expression profiling. Rather than functioning as a switch, Bvg controls a remarkable continuum of gene expression states, with hundreds of genes maximally expressed in intermediate phases between the Bvg(+) and Bvg(-) poles. Comparative analysis of Bvg regulation in B. pertussis and B. bronchiseptica revealed a relatively conserved Bvg(+) phase transcriptional program and identified previously uncharacterized candidate virulence factors. In contrast, control of Bvg(-)- and Bvg(i)-phase genes diverged substantially between species; regulation of metabolic, transporter, and motility loci indicated an increased capacity in B. bronchiseptica, compared to B. pertussis, for ex vivo adaptation. Strain comparisons also demonstrated variation in gene expression patterns within species. Among the genes with the greatest variability in patterns of expression, predicted promoter sequences were nearly identical. Our data suggest that the complement of transcriptional regulators is largely responsible for transcriptional diversity. In support of this hypothesis, many putative transcriptional regulators that were Bvg regulated in B. bronchiseptica were deleted, inactivated, or unregulated by BvgAS in B. pertussis. We propose the concept of a "flexible regulon." This flexible regulon may prove to be important for pathogen evolution and the diversification of host range specificity.

Characterization of a highly conserved island in the otherwise divergent Bordetella holmesii and Bordetella pertussis genomes.

The recently discovered pathogen Bordetella holmesii has been isolated from the airways and blood of diseased humans. Genetic events contributing to the emergence of B. holmesii are not understood, and its phylogenetic position among the bordetellae remains unclear. To address these questions, B. holmesii strains were analyzed by comparative genomic hybridization (CGH) to a Bordetella pertussis microarray and by multilocus sequence typing. Both methods indicated substantial sequence divergence between B. pertussis and B. holmesii. However, CGH identified a putative pathogenicity island of 66 kb that is highly conserved between these species and contains several IS481 elements that may have been laterally transferred from B. pertussis to B. holmesii. This island contains, among other genes, a functional, iron-regulated locus encoding the biosynthesis, export, and uptake of the siderophore alcaligin. The acquisition of this genomic island by B. holmesii may have significantly contributed to its emergence as a human pathogen. Horizontal gene transfer between B. pertussis and B. holmesii may also explain the unusually high sequence identity of their 16S rRNA genes.

The identification of uncultured microbial pathogens.

Clinicians have long been aware of human diseases that are associated with visible but uncultured microorganisms. Without the ability to cultivate these organisms, they have remained unidentified. Environmental microbiologists have also discovered on the basis of recent advances in the field of molecular phylogeny that culture-based methods for detecting microorganisms are biased and insensitive. A culture-independent experimental approach is described for the identification of microbial pathogens. This approach incorporates fundamental aspects of 16S rRNA-based molecular phylogeny as well as nucleic acid amplification technology. From its application to Whipple's disease, one can speculate as to the potential insights a highly sensitive, culture-independent method may provide into the diversity and natural ecology of human microbial pathogens.

Detection and identification of previously unrecognized microbial pathogens.

Features of a number of important but poorly explained human clinical syndromes strongly indicate a microbial etiology. In these syndromes, the failure of cultivation-dependent microbial detection methods reveals our ignorance of microbial growth requirements. Sequence-based molecular methods, however, offer alternative approaches for microbial identification directly from host specimens found in the setting of unexplained acute illnesses, chronic inflammatory disease, and from anatomic sites that contain commensal microflora. The rapid expansion of genome sequence databases and advances in biotechnology present opportunities and challenges: identification of consensus sequences from which reliable, specific phylogenetic information can be inferred for all taxonomic groups of pathogens, broad-range pathogen identification on the basis of virulence-associated gene families, and use of host gene expression response profiles as specific signatures of microbial infection.

Identification of uncultured microorganisms: expanding the spectrum of characterized microbial pathogens.

The combination of enzymatic nucleic acid amplification techniques with 16S rRNA-based molecular phylogeny has brought about a new approach to the identification of microbial pathogens that can not be cultivated in the laboratory. The applications of this experimental approach to bacillary angiomatosis and to Whipple's disease have revealed the presence of two previously uncharacterized organisms. These results suggest the existence of a far greater microbial diversity among human pathogens than has been so far appreciated with culture-dependent methods. PCR-based studies of aquatic environmental microbial communities have already reached similar conclusions. As a result, new and provocative questions are raised concerning the association of amplified 16S rRNA sequences with diseased tissue. The answers must await the results of further investigations and the expansion of sequence data bases.

Using DNA microarrays to study host-microbe interactions.

Complete genomic sequences of microbial pathogens and hosts offer sophisticated new strategies for studying host-pathogen interactions. DNA microarrays exploit primary sequence data to measure transcript levels and detect sequence polymorphisms, for every gene, simultaneously. The design and construction of a DNA microarray for any given microbial genome are straightforward. By monitoring microbial gene expression, one can predict the functions of uncharacterized genes, probe the physiologic adaptations made under various environmental conditions, identify virulence-associated genes, and test the effects of drugs. Similarly, by using host gene microarrays, one can explore host response at the level of gene expression and provide a molecular description of the events that follow infection. Host profiling might also identify gene expression signatures unique for each pathogen, thus providing a novel tool for diagnosis, prognosis, and clinical management of infectious disease.