CHARACTERIZATION OF THE FUNGAL MICROBIOTA IN THE NOSTRILS AND RECTUM OF AMAZONIAN MANATEES (TRICHECHUS INUNGUIS) FROM A REHABILITATION PROGRAM IN BRAZIL.

The present study characterized the filamentous and yeast-like fungal microbiota of the nasal cavity and rectum of Amazonian manatees (Trichechus inunguis) undergoing rehabilitation at the Laboratory of Aquatic Mammals, National Institute of Amazonian Research, Manaus, Amazonas, and determined the antifungal susceptibility of these organisms. Nasal and rectal swabs were collected from 22 calves and three juveniles. The samples were seeded in Sabouraud agar supplemented with chloramphenicol 10%, incubated at 26°C, and observed daily for up to 7 d. The growth of different filamentous and yeast-like fungi was observed among the two anatomical sites. Filamentous fungi were categorized by macro- and microscopic characteristics of the colonies. Representatives of each group were selected for molecular identification based on the internal transcribed spacer region. Yeast identification was performed using MALDI-TOF MS and molecular analyses. Thirteen genera of filamentous fungi and six genera of yeasts were isolated and identified. The dominant filamentous species were Fusarium spp., Aspergillus spp., and Cochliobolus lunatus in the nostril samples and Aspergillus melleus in the rectal samples. Candida was the dominant genus among the identified yeasts at both anatomical sites. In the antifungal susceptibility test, 28 isolates showed resistance to fluconazole (78%), itraconazole (39%), and nystatin (42%). The knowledge of fungal microbiota composition of Amazonian manatees provides information that assists in monitoring the health status of individuals maintained in captivity, as these organisms can behave either as opportunists or as primary pathogens. Moreover, the composition and resistance of these organisms may vary among different rehabilitation institutions or different time frames of search, reinforcing the importance of constant in loco surveillance of these microorganisms. This study provides new perspectives on the fungal diversity in the microbiota of manatees and supports future studies concerning the clinical and epidemiological aspects and the impacts of these agents on the health of Amazonian manatees undergoing rehabilitation.

Severe outbreak of Centrocestus formosanus (Trematoda: Heterophyidae) in farm-raised ornamental platies Xiphophorus maculatus.

This report describes a severe outbreak of the gill fluke Centrocestus formosanus in farm-raised platies Xiphophorus maculatus in Brazil, with mortality rate approaching 95%. Typical clinical signs of infection were observed, with microscopic examinations of fresh gills revealing multiple cysts containing a once-folded metacercaria with an X-shaped excretory bladder. The 18S subunit of the metacercariae (BR1) was amplified by PCR, sequenced and analyzed by BLAST. Subsequent phylogenetic analyses revealed that the BR1 isolate was closely related to C. formosanus from Thailand. This is the first report of C. formosanus in ornamental fish in Brazil. Our observations suggest that platies are highly sensitive to this digenetic parasite. Controlling population densities of the parasite's intermediate host, the snail Melanoides tuberculata, would help to reduce outbreaks.

First genome sequencing and comparative analyses of Corynebacterium pseudotuberculosis strains from Mexico.

Corynebacterium pseudotuberculosis is a pathogenic bacterium which has been rapidly spreading all over the world, causing economic losses in the agricultural sector and sporadically infecting humans. Six C. pseudotuberculosis strains were isolated from goats, sheep, and horses with distinct abscess locations. For the first time, Mexican genomes of this bacterium were sequenced and studied in silico. All strains were sequenced using Ion Personal Genome Machine sequencer, assembled using Newbler and SPAdes software. The automatic genome annotation was done using the software RAST and in-house scripts for transference, followed by manual curation using Artemis software and BLAST against NCBI and UniProt databases. The six genomes are publicly available in NCBI database. The analysis of nucleotide sequence similarity and the generated phylogenetic tree led to the observation that the Mexican strains are more similar between strains from the same host, but the genetic structure is probably more influenced by transportation of animals between farms than host preference. Also, a putative drug target was predicted and in silico analysis of 46 strains showed two gene clusters capable of differentiating the biovars equi and ovis: Restriction Modification system and CRISPR-Cas cluster.

Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms.

Streptococcus agalactiae is a major pathogen and a hindrance on tilapia farming worldwide. The aims of this work were to analyze the genomic evolution of Brazilian strains of S. agalactiae and to establish spatial and temporal relations between strains isolated from different outbreaks of streptococcosis. A total of 39 strains were obtained from outbreaks and their whole genomes were sequenced and annotated for comparative analysis of multilocus sequence typing, genomic similarity and whole genome multilocus sequence typing (wgMLST). The Brazilian strains presented two sequence types, including a newly described ST, and a non-typeable lineage. The use of wgMLST could differentiate each strain in a single clone and was used to establish temporal and geographical correlations among strains. Bayesian phylogenomic analysis suggests that the studied Brazilian population was co-introduced in the country with their host, approximately 60 years ago. Brazilian strains of S. agalactiae were shown to be heterogeneous in their genome sequences and were distributed in different regions of the country according to their genotype, which allowed the use of wgMLST analysis to track each outbreak event individually.

Quantitative Proteomic Analysis Reveals Changes in the Benchmark Corynebacterium pseudotuberculosis Biovar Equi Exoproteome after Passage in a Murine Host.

Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.

A shift in the virulence potential of Corynebacterium pseudotuberculosis biovar ovis after passage in a murine host demonstrated through comparative proteomics.

BACKGROUND: Corynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage. RESULTS: The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen. CONCLUSIONS: This study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.

Complete Genome Sequence of the Attenuated Corynebacterium pseudotuberculosis Strain T1.

We present here the genome sequence of the attenuated Corynebacterium pseudotuberculosis strain T1. The sequencing was performed with an Ion Torrent Personal Genome Machine platform. The genome is a circular chromosome of 2,337,201 bp, with a G+C content of 52.85% and a total of 2,125 coding sequences (CDSs), 12 rRNAs, 49 tRNAs, and 24 pseudogenes.

Evaluating the efficacy of the new Ion PGM Hi-Q Sequencing Kit applied to bacterial genomes.

Benchtop NGS platforms are constantly evolving to follow new advances in genomics. Thus, the manufacturers are making improvements, such as the recent Ion PGM Hi-Q chemistry. We evaluate the efficacy of this new Hi-Q approach by comparing it with the former Ion PGM kit and the Illumina MiSEQ Nextera 3rd version. The Hi-Q chemistry showed improvement on mapping reads, with 49 errors for 10kbp mapped; in contrast, the former kit had 89 errors. Additionally, there was a reduction of 80% in erroneous variant detection with the Torrent Variant Caller. Also, an enhancement was observed in de novo assembly with a more confident result in whole-genome MLST, with up to 96% of the alleles assembled correctly for both tested microbial genomes. All of these advantages result in a final genome sequence closer to the performance with MiSEQ and will contribute to turn comparative genomic analysis a reliable task.

Complete Genome Sequence of Corynebacterium pseudotuberculosis Viscerotropic Strain N1.

We present the complete genome sequence of Corynebacterium pseudotuberculosis strain N1. The sequencing was performed with the Ion Torrent Personal Genome Machine system. The genome is a circular chromosome with 2,337,845 bp, a G+C content of 52.85%, and a total of 2,045 coding sequences, 12 rRNAs, 49 tRNAs, and 58 pseudogenes.

Complete genome sequence of Peptoclostridium difficile strain Z31.

BACKGROUND: Peptoclostridium (Clostridium) difficile is a spore-forming bacterium responsible for nosocomial infections in humans. It is recognized as an important agent of diarrhea and colitis in several animal species and a possible zoonotic agent. Despite the known importance of P. difficile infection in humans and animals, no vaccine or other effective measure to control the disease is commercially available. A possible alternative treatment for P. difficile infection is the use of a nontoxigenic strain of P. difficile as a competitive exclusion agent. However, a thorough knowledge of this strain is necessary for this purpose. We selected P. difficile Z31, a nontoxigenic strain (PCR ribotype 009), for investigation because it prevents P. difficile infection in a hamster model. RESULTS: The genome sequence of P. difficile Z31 is a circular chromosome of 4298,263 bp, with a 29.21 % GC content, encoding 4128 proteins, and containing 78 pseudogenes. This strain belongs to ST 3, clade 1, and has five phage regions in its genome. Genes responsible for resistance to tetracycline and erythromycin were detected and more importantly, Z31 also contains genes that promote spore production and stability, cell attachment, intestinal adherence, and biofilm formation. CONCLUSION: In this study, we present the first complete genome sequence of nontoxigenic P. difficile strain Z31. When the Z31 genome was compared with those of other isolates available in GenBank, including a draft genome of a nontoxigenic strain, several unique regions were evident. Z31 contains no toxin genes, but encodes several non-toxin virulence factors, which may favor host colonization.

Comparative genome analysis of Weissella ceti, an emerging pathogen of farm-raised rainbow trout.

BACKGROUND: The genus Weissella belongs to the lactic acid bacteria and includes 18 currently identified species, predominantly isolated from fermented food but rarely from cases of bacteremia in animals. Recently, a new species, designated Weissella ceti, has been correlated with hemorrhagic illness in farm-raised rainbow trout in China, Brazil, and the USA, with high transmission and mortality rates during outbreaks. Although W. ceti is an important emerging veterinary pathogen, little is known about its genomic features or virulence mechanisms. To better understand these and to characterize the species, we have previously sequenced the genomes of W. ceti strains WS08, WS74, and WS105, isolated from different rainbow trout farms in Brazil and displaying different pulsed-field gel electrophoresis patterns. Here, we present a comparative analysis of the three previously sequenced genomes of W. ceti strains from Brazil along with W. ceti NC36 from the USA and those of other Weissella species. RESULTS: Phylogenomic and orthology-based analyses both showed a high-similarity in the genetic structure of these W. ceti strains. This structure is corroborated by the highly syntenic order of their genes and the neutral evolution inferred from Tajima's D. A whole-genome multilocus sequence typing analysis distinguished strains WS08 and NC36 from strains WS74 and WS105. We predicted 10 putative genomic islands (GEI), among which PAIs 3a and 3b are phage sequences that occur only in WS105 and WS74, respectively, whereas PAI 1 is species specific. CONCLUSIONS: We identified several genes putatively involved in the basic processes of bacterial physiology and pathogenesis, including survival in aquatic environment, adherence in the host, spread inside the host, resistance to immune-system-mediated stresses, and antibiotic resistance. These data provide new insights in the molecular epidemiology and host adaptation for this emerging pathogen in aquaculture.

Draft Genome Sequences of Two Species of "Difficult-to-Identify" Human-Pathogenic Corynebacteria: Implications for Better Identification Tests.

Non-diphtheriae Corynebacterium species have been increasingly recognized as the causative agents of infections in humans. Differential identification of these bacteria in the clinical microbiology laboratory by the most commonly used biochemical tests is challenging, and normally requires additional molecular methods. Herein, we present the annotated draft genome sequences of two isolates of "difficult-to-identify" human-pathogenic corynebacterial species: C. xerosis and C. minutissimum. The genome sequences of ca. 2.7 Mbp, with a mean number of 2,580 protein encoding genes, were also compared with the publicly available genome sequences of strains of C. amycolatum and C. striatum. These results will aid the exploration of novel biochemical reactions to improve existing identification tests as well as the development of more accurate molecular identification methods through detection of species-specific target genes for isolate's identification or drug susceptibility profiling.

Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis.

BACKGROUND: Caseous lymphadenitis (CLA) is an infectious disease that affects small ruminants and is caused by Corynebacterium pseudotuberculosis. This disease is responsible for high economic losses due to condemnation and trim of infected carcasses, decreased leather and wool yield, loss of sales of breeding stock and deaths from internal involvement. Treatment is costly and ineffective; the most cost-effective strategy is timely immunisation. Various vaccine strategies have been tested, and recombinant vaccines are a promising alternative. Thus, in this study, different vaccine formulations using a recombinant protein (rCP40) and the CP09 live recombinant strain were evaluated. Five groups of 10 mice each were immunised with saline (G1), rCP40 (G2), CP09 (G3), a combination of CP09 and rCP40 (G4) and a heterologous prime-boost strategy (G5). Mice received two immunisations within 15 days. On day 30 after primary immunisation, all groups were challenged with a C. pseudotuberculosis virulent strain. Mice were monitored and mortality was recorded for 30 days after challenge. RESULTS: The G2, G4 and G5 groups showed high levels of IgG1 and IgG2a; G2 presented significant IgG2a production after virulent challenge in the absence of IgG1 and IgG3 induction. Thirty days after challenge, the mice survival rates were 20 (G1), 90 (G2), 50 (G3), 70 (G4) and 60% (G5). CONCLUSIONS: rCP40 is a promising target in the development of vaccines against caseous lymphadenitis.

Genome Sequence of Lactococcus lactis subsp. lactis NCDO 2118, a GABA-Producing Strain.

Lactococcus lactis subsp. lactis NCDO 2118 is a nondairy lactic acid bacterium, a xylose fermenter, and a gamma-aminobutyric acid (GABA) producer isolated from frozen peas. Here, we report the complete genome sequence of L. lactis NCDO 2118, a strain with probiotic potential activity.

Identification of 11 new exoproteins in Corynebacterium pseudotuberculosis by comparative analysis of the exoproteome.

This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT(®). A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.

The pan-genome of the animal pathogen Corynebacterium pseudotuberculosis reveals differences in genome plasticity between the biovar ovis and equi strains.

Corynebacterium pseudotuberculosis is a facultative intracellular pathogen and the causative agent of several infectious and contagious chronic diseases, including caseous lymphadenitis, ulcerative lymphangitis, mastitis, and edematous skin disease, in a broad spectrum of hosts. In addition, Corynebacterium pseudotuberculosis infections pose a rising worldwide economic problem in ruminants. The complete genome sequences of 15 C. pseudotuberculosis strains isolated from different hosts and countries were comparatively analyzed using a pan-genomic strategy. Phylogenomic, pan-genomic, core genomic, and singleton analyses revealed close relationships among pathogenic corynebacteria, the clonal-like behavior of C. pseudotuberculosis and slow increases in the sizes of pan-genomes. According to extrapolations based on the pan-genomes, core genomes and singletons, the C. pseudotuberculosis biovar ovis shows a more clonal-like behavior than the C. pseudotuberculosis biovar equi. Most of the variable genes of the biovar ovis strains were acquired in a block through horizontal gene transfer and are highly conserved, whereas the biovar equi strains contain great variability, both intra- and inter-biovar, in the 16 detected pathogenicity islands (PAIs). With respect to the gene content of the PAIs, the most interesting finding is the high similarity of the pilus genes in the biovar ovis strains compared with the great variability of these genes in the biovar equi strains. Concluding, the polymerization of complete pilus structures in biovar ovis could be responsible for a remarkable ability of these strains to spread throughout host tissues and penetrate cells to live intracellularly, in contrast with the biovar equi, which rarely attacks visceral organs. Intracellularly, the biovar ovis strains are expected to have less contact with other organisms than the biovar equi strains, thereby explaining the significant clonal-like behavior of the biovar ovis strains.

Genome sequence of Corynebacterium pseudotuberculosis biovar equi strain 258 and prediction of antigenic targets to improve biotechnological vaccine production.

Corynebacterium pseudotuberculosis is the causative agent of several veterinary diseases in a broad range of economically important hosts, which can vary from caseous lymphadenitis in sheep and goats (biovar ovis) to ulcerative lymphangitis in cattle and horses (biovar equi). Existing vaccines against C. pseudotuberculosis are mainly intended for small ruminants and, even in these hosts, they still present remarkable limitations. In this study, we present the complete genome sequence of C. pseudotuberculosis biovar equi strain 258, isolated from a horse with ulcerative lymphangitis. The genome has a total size of 2,314,404 bp and contains 2088 predicted protein-coding regions. Using in silico analysis, eleven pathogenicity islands were detected in the genome sequence of C. pseudotuberculosis 258. The application of a reverse vaccinology strategy identified 49 putative antigenic proteins, which can be used as candidate vaccine targets in future works.

Progression of 'OMICS' methodologies for understanding the pathogenicity of Corynebacterium pseudotuberculosis: the Brazilian experience.

Since the first successful attempt at sequencing the Corynebacterium pseudotuberculosis genome, large amounts of genomic, transcriptomic and proteomic data have been generated. C. pseudotuberculosis is an interesting bacterium due to its great zoonotic potential and because it causes considerable economic losses worldwide. Furthermore, different strains of C. pseudotuberculosis are capable of causing various diseases in different hosts. Currently, we seek information about the phylogenetic relationships between different strains of C. pseudotuberculosis isolates from different hosts across the world and to employ these data to develop tools to diagnose and eradicate the diseases these strains cause. In this review, we present the latest findings on C. pseudotuberculosis that have been obtained with the most advanced techniques for sequencing and genomic organization. We also discuss the development of in silico tools for processing these data to prompt a better understanding of this pathogen.

Complete genome sequences of Corynebacterium pseudotuberculosis strains 3/99-5 and 42/02-A, isolated from sheep in Scotland and Australia, respectively.

Here, we report the whole-genome sequences of two ovine-pathogenic Corynebacterium pseudotuberculosis isolates: strain 3/99-5, which represents the first C. pseudotuberculosis genome originating from the United Kingdom, and 42/02-A, the second from Australia. These genome sequences will contribute to the objective of determining the global pan-genome of this bacterium.

Complete genome sequence of Corynebacterium pseudotuberculosis strain 1/06-A, isolated from a horse in North America.

Corynebacterium pseudotuberculosis causes disease in several animal species, although distinct biovars exist that appear to be restricted to specific hosts. In order to facilitate a better understanding of the differences between biovars, we report here the complete genome sequence of the equine pathogen Corynebacterium pseudotuberculosis strain 1/06-A.