Delineation of the pan-proteome of fish-pathogenic Streptococcus agalactiae strains using a label-free shotgun approach.

BACKGROUND: Streptococcus agalactiae (GBS) is a major pathogen of Nile tilapia, a global commodity of the aquaculture sector. The aims of this study were to evaluate protein expression in the main genotypes of GBS isolated from diseased fishes in Brazil using a label-free shotgun nano-liquid chromatography-ultra definition mass spectrometry (nanoLC-UDMSE) approach and to compare the differential abundance of proteins identified in strains isolated from GBS-infected fishes and humans. RESULTS: A total of 1070 protein clusters were identified by nanoLC-UDMSE in 5 fish-adapted GBS strains belonging to sequence types ST-260 and ST-927 and the non-typeable (NT) lineage and 1 human GBS strain (ST-23). A total of 1065 protein clusters corresponded to the pan-proteome of fish-adapted GBS strains; 989 of these were identified in all fish-adapted GBS strains (core proteome), and 62 were shared by at least two strains (accessory proteome). Proteins involved in the stress response and in the regulation of gene expression, metabolism and virulence were detected, reflecting the adaptive ability of fish-adapted GBS strains in response to stressor factors that affect bacterial survival in the aquatic environment and bacterial survival and multiplication inside the host cell. Measurement of protein abundance among different hosts showed that 5 and 26 proteins were exclusively found in the human- and fish-adapted GBS strains, respectively; the proteins exclusively identified in fish isolates were mainly related to virulence factors. Furthermore, 215 and 269 proteins were up- and down-regulated, respectively, in the fish-adapted GBS strains in comparison to the human isolate. CONCLUSIONS: Our study showed that the core proteome of fish-adapted GBS strains is conserved and demonstrated high similarity of the proteins expressed by fish-adapted strains to the proteome of the human GBS strain. This high degree of proteome conservation of different STs suggests that, a monovalent vaccine may be effective against these variants.

Effects of temperature changes in the transcriptional profile of the emerging fish pathogen Francisella noatunensis subsp. orientalis.

One of the major challenges in Nile tilapia (Oreochromis niloticus L.) farming is the occurrence of bacterial infections, and the Francisella noatunensis subsp. orientalis (FNO) is an important pathogen that has emerged in last decades. Francisellosis outbreaks have been reported in the literature as occurring seasonally when water temperature is below 24 °C. The aim of this study was to quantify the median lethal doses (LD50) of FNO in experimental challenges at 28 °C and 22 °C, and to investigate the impact of temperature changes in whole genome expression using microarray technology. The LD50 for Nile tilapia at 28 °C was ∼105.7, whereas at 22 °C, the LD50 was ∼102.2, showing that the decrease in temperature enhanced disease outcome. Out of 1917 genes screened, a total of 31 and 19 genes were down- and up-regulated at 22 °C, respectively. These genes were grouped by orthology into functional categories of: amino acid, inorganic ion, and carbohydrate transport and metabolism; transcription; and posttranslational modification, protein turnover, and chaperones. Expression of genes related to metabolism, oxidative stress, and thermal shock were regulated by temperature changes, reflecting an ability of FNO to adapt to the environment. Expression of virulence genes usually required for the Francisella genus was not changed between tested temperatures, including that of genes located on the Francisella Pathogenicity Island.

Complete genome sequencing of sixteen Francisella noatunensis subsp. orientalis isolates: A genomic approach for molecular characterization and spread dynamics of this clonal population.

Francisella noatunensis subsp. orientalis (FNO) is an important emerging pathogen associated with disease outbreaks in farm-raised Nile tilapia. FNO genetic diversity using PCR-based typing, no intra-species discrimination was achieved among isolates/strains from different countries, thus demonstrating a clonal behaviour pattern. In this study, we aimed to evaluate the population structure of FNO isolates by comparing whole-genome sequencing data. The analysis of recombination showed that Brazilian isolates group formed a clonal population; whereas other lineages are also supported by this analysis for isolates from foreign countries. The whole-genome multilocus sequence typing (wgMLST) analysis showed varying numbers of dissimilar alleles, suggesting that the Brazilian clonal population are in expansion. Each Brazilian isolate could be identified as a single node by high-resolution gene-by-gene approach, presenting slight genetic differences associated to mutational events. The common ancestry node suggests a single entry into the country before 2012, and the rapid dissemination of this infectious agent may be linked to market sales of infected fingerlings.

Whole-genome optical mapping reveals a mis-assembly between two rRNA operons of Corynebacterium pseudotuberculosis strain 1002.

BACKGROUND: Studies have detected mis-assemblies in genomes of the species Corynebacterium pseudotuberculosis. These new discover have been possible due to the evolution of the Next-Generation Sequencing platforms, which have provided sequencing with accuracy and reduced costs. In addition, the improving of techniques for construction of high accuracy genomic maps, for example, Whole-genome mapping (WGM) (OpGen Inc), have allow high-resolution assembly that can detect large rearrangements. RESULTS: In this work, we present the resequencing of Corynebacterium pseudotuberculosis strain 1002 (Cp1002). Cp1002 was the first strain of this species sequenced in Brazil, and its genome has been used as model for several studies in silico of caseous lymphadenitis disease. The sequencing was performed using the platform Ion PGM and fragment library (200 bp kit). A restriction map was constructed, using the technique of WGM with the enzyme KpnI. After the new assembly process, using WGM as scaffolder, we detected a large inversion with size bigger than one-half of genome. A specific analysis using BLAST and NR database shows that the inversion occurs between two homology RNA ribosomal regions. CONCLUSION: In conclusion, the results showed by WGM could be used to detect mismatches in assemblies, providing genomic maps with high resolution and allow assemblies with more accuracy and completeness. The new assembly of C. pseudotuberculosis was deposited in GenBank under the accession no. CP012837.

First report of infectious spleen and kidney necrosis virus in Nile tilapia in Brazil.

In June 2020, an atypical fatal outbreak in a Brazilian Nile tilapia farm was investigated. Twenty-three animals were collected and different tissues were used for bacterial isolation, histopathological and electron microscopic examination and viral detection using molecular methods. A large number of megalocytes were observed in the histopathological analysis of several tissues. Icosahedral virions, with a diameter of approximately 160 nm, were visualized inside the megalocytes through transmission electron microscopy of the spleen tissue. The virions were confirmed to be infectious spleen and kidney necrosis virus (ISKNV) through PCR and sequencing analyses of the fish samples. Phylogenetic analysis indicated that the virus belongs to the Clade 1 of ISKNV. This viral pathogen is associated with high mortality in the early stages of cultured Nile tilapia in the United States, Thailand and Ghana; however, until now, there have been no reports from ISKNV affecting cultured fish in Brazil. Additionally, in 14 out of 23 sampled fish, Streptococcus agalactiae, Edwardsiella tarda or Aeromonas hydrophila infections were also detected. This is the first report of fatal ISKNV infections in the Brazilian Nile tilapia fish farms and represents a new challenge to the aquaculture sector in the country.

Transmission of Escherichia coli Causing Pyometra between Two Female Dogs.

Despite its clinical relevance, the pathogenesis of canine pyometra remains poorly understood. To date, it is recognized as a non-transmissible infectious disease. In this study, the simultaneous occurrence of pyometra and Escherichia coli in two cohabitant female dogs underwent in-depth investigation due to the hypothesis of transmission between these animals. Two 5-year-old Chow Chow dogs (namely, dogs 23 and 24-D23 and D24) were referred to a veterinary hospital with suspected pyometra. Both animals showed prostration, anorexia, and purulent vulvar discharge over a 1-week period. After ovariohysterectomy, uterine tissue, uterine contents, and rectal swabs were collected for histopathological and microbiological analysis. Uterine histology demonstrated purulent material and multifocal necrosis with endometrial ulceration, and a morphological diagnosis of pyometra was confirmed. Furthermore, E. coli from the same phylogroup (B2) and positive for the same virulence factors with the same antimicrobial susceptibility profile was isolated from the uterine contents of both dogs and the rectum of D23. Conversely, the E. coli strains recovered from D24 differed in phylogroup (one isolate), virulence factors (all three isolates), and antimicrobial susceptibility (all three isolates). Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) suggested that all isolates from the uterine content of both dogs and the rectal swab of D23 were 100% the same, but different from all isolates in the rectal swab of D24. One isolate from the uterine content of each animal as well as rectal swabs were subjected to whole-genome sequencing (WGS). Both whole-genome multilocus sequence typing(wgMLST) and single-nucleotide polymorphism (SNP) analysis supported the hypothesis that the isolates from the uterine content of both animals and the rectal swab of D23 were clonal. Taken together, these clinical features, pathology, microbiology, and molecular findings suggest, to the best of our knowledge, the first transmission of E. coli associated with pyometra between two animals. These results could impact the management of sites where several females cohabit in the same local area such as kennels.

Comparative polyphasic characterization of Weissella strains isolated from beaked whale and rainbow trout (Oncorhynchus mykiss): confirmation of Weissella ceti sp. nov. and description of the novel Weissella tructae sp. nov. isolated from farmed rainbow trout.

The weissellosis agent bacterium (WS08T = CBMAI 2730) was isolated from diseased rainbow trout (Oncorhynchus mykiss) in Brazil. The whole genome sequence of this strain was compared with the Mexican W-1 strain, also isolated from diseased rainbow trout, and with the Weissella ceti type strain CECT 7719 T (= 1119-1A-09 T = CCUG 59653 T), recovered from the beaked whale. Digital DNA-DNA hybridization pairwise analyses scored 98.7% between the Mexican W-1 and Brazilian WS08T but just 24.4% for both fish isolates compared to the W. ceti type strain CECT 7719 T. The 16S rRNA gene sequence comparisons with isolates of W. ceti, available at GenBank, were conducted. All rainbow trout-pathogenic isolates grouped close (97% bootstrap confirmation), but when this group was compared to the W. ceti type strain CECT 7719 T the similarity varied from 78.9 to 79.1%. Phenotypic assays were also conducted, and the W. ceti type strain diverged from WS08T and W-1 in the hydrolysis of aesculin, D-mannose, and potassium gluconate and in the hydrolysis of hippurate. Moreover, WS08T and W-1 showed weak growth at 5 °C whereas no growth was observed for W. ceti CECT 7719 T. The major fatty acids (> 10% total fatty acids) presented by WS08T and W-1 were summed feature 8 (C18:1 ω7c/C18:1 ω6c), summed feature 3 (C16:1 ω6c/C16:1ω7c), and C16:0. The results of phylogenetic and phenotypic analyses clearly differentiated the W. ceti CECT 7719 T type strain from the assessed pathogenic strains obtained from rainbow trout. Therefore, Weissella strains isolated from rainbow trout, here represented by strain WS08T (= CBMAI 2730), should be known as members of a novel species for which the name Weissella tructae sp. nov. is proposed.

Re-sequencing and optical mapping reveals misassemblies and real inversions on Corynebacterium pseudotuberculosis genomes.

The number of draft genomes deposited in Genbank from the National Center for Biotechnology Information (NCBI) is higher than the complete ones. Draft genomes are assemblies that contain fragments of misassembled regions (gaps). Such draft genomes present a hindrance to the complete understanding of the biology and evolution of the organism since they lack genomic information. To overcome this problem, strategies to improve the assembly process are developed continuously. Also, the greatest challenge to the assembly progress is the presence of repetitive DNA regions. This article highlights the use of optical mapping, to detect and correct assembly errors in Corynebacterium pseudotuberculosis. We also demonstrate that choosing a reference genome should be done with caution to avoid assembly errors and loss of genetic information.

Gut microbiome modulation during treatment of mucositis with the dairy bacterium Lactococcus lactis and recombinant strain secreting human antimicrobial PAP.

Mucositis is an inflammatory condition of the gut, caused by an adverse effect of chemotherapy drugs, such as 5-fluorouracil (5-FU). In an attempt to develop alternative treatments for the disease, several research groups have proposed the use of probiotics, in particular, Lactic Acid Bacteria (LAB). In this context, the use of recombinant LAB, for delivering anti-inflammatory compounds has also been explored. In previous work, we demonstrated that either Lactococcus lactis NZ9000 or a recombinant strain expressing an antimicrobial peptide involved in human gut homeostasis, the Pancreatitis-associated Protein (PAP), could ameliorate 5-FU-induced mucositis in mice. However, the impact of these strains on the gut microbiota still needs to be elucidated. Therefore, in the present study, we aimed to characterize the effects of both Lactococci strains in the gut microbiome of mice through a 16 S rRNA gene sequencing metagenomic approach. Our data show 5-FU caused a significant decrease in protective bacteria and increase of several bacteria associated with pro-inflammatory traits. The Lactococci strains were shown to reduce several potential opportunistic microbes, while PAP delivery was able to suppress the growth of Enterobacteriaceae during inflammation. We conclude the strain secreting antimicrobial PAP was more effective in the control of 5-FU-dysbiosis.

Comparative genomic analysis between Corynebacterium pseudotuberculosis strains isolated from buffalo.

Corynebacterium pseudotuberculosis is a Gram-positive, pleomorphic, facultative intracellular pathogen that causes Oedematous Skin Disease (OSD) in buffalo. To better understand the pathogenic mechanisms of OSD, we performed a comparative genomic analysis of 11 strains of C. pseudotuberculosis isolated from different buffalo found to be infected in Egypt during an outbreak that occurred in 2008. Sixteen previously described pathogenicity islands (PiCp) were present in all of the new buffalo strains, but one of them, PiCp12, had an insertion that contained both a corynephage and a diphtheria toxin gene, both of which may play a role in the adaptation of C. pseudotuberculosis to this new host. Synteny analysis showed variations in the site of insertion of the corynephage during the same outbreak. A gene functional comparison showed the presence of a nitrate reductase operon that included genes involved in molybdenum cofactor biosynthesis, which is necessary for a positive nitrate reductase phenotype and is a possible adaptation for intracellular survival. Genomes from the buffalo strains also had fusions in minor pilin genes in the spaA and spaD gene cluster (spaCX and spaYEF), which could suggest either an adaptation to this particular host, or mutation events in the immediate ancestor before this particular epidemic. A phylogenomic analysis confirmed a clear separation between the Ovis and Equi biovars, but also showed what appears to be a clustering by host species within the Equi strains.

Complete genome sequences of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190: a fish pathogen with genomic clonal behavior.

The genus Francisella is composed of Gram-negative, pleomorphic, strictly aerobic and non-motile bacteria, which are capable of infecting a variety of terrestrial and aquatic animals, among which Francisella noatunensis subsp. orientalis stands out as the causative agent of pyogranulomatous and granulomatous infections in fish. Accordingly, F. noatunensis subsp. orientalis is responsible for high mortality rates in freshwater fish, especially Nile Tilapia. In the current study, we present the genome sequences of F. noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190. The genomes include one circular chromosome of 1,859,720 bp, consisting of 32 % GC content, 1538 coded proteins and 363 pseudogenes for FNO12; one circular chromosome of 1,862,322 bp, consisting of 32 % GC content, 1537 coded proteins and 365 pseudogenes for FNO24; and one circular chromosome of 1,859,595 bp, consisting of 32 % GC content, 1539 coded proteins and 362 pseudogenes for FNO190. All genomes have similar genetic content, implicating a clonal-like behavior for this species.

Whole-Genome Sequence of Corynebacterium auriscanis Strain CIP 106629 Isolated from a Dog with Bilateral Otitis from the United Kingdom.

In this work, we describe a set of features of Corynebacterium auriscanis CIP 106629 and details of the draft genome sequence and annotation. The genome comprises a 2.5-Mbp-long single circular genome with 1,797 protein-coding genes, 5 rRNA, 50 tRNA, and 403 pseudogenes, with a G+C content of 58.50%.

Draft Genome Sequences of Two Pathogenic Corynebacterial Species Isolated from Cows.

The species Corynebacterium renale, Corynebacterium pilosum, and Corynebacterium cystitidis were initially thought to be the same species C. renale, but with different immunological types. These bacteria are the causative agent of cystitis, urethritis and pyelonephritis and are found usually as constituents of the normal flora in the lower urogenital tract of cattle. Therefore, we present the draft genome sequences of two pathogenic Corynebacterium species: C. renale CIP 52.96 and C. pilosum CIP 103422. The genome sequences of these species have 2,322,762 bp with 2,218 protein encoding genes and 2,548,014 bp with 2,428 protein encoding genes, respectively. These genomes can help clarify the virulence mechanisms of these unknown bacteria and enable the development of more effective methods for control.

The genome anatomy of Corynebacterium pseudotuberculosis VD57 a highly virulent strain causing Caseous lymphadenitis.

Corynebacterium pseudotuberculosis strain VD57 (Cp_VD57), a highly virulent, nonmotile, non-sporulating, and a mesophilic bacterium, was isolated from a goat's granulomatous lesion in the municipality of Juazeiro, Bahia State, Brazil. Here, we describe a set of features of the strain, together with the details of its complete genome sequence and annotation. The genome comprises of a 2.5 Mbp long, single circular genome with 2,101 protein-coding genes, 12 rRNA, 49 tRNA and 47 pseudogenes and a G + C content of 52.85 %. Genetic variation was detected in Cp_VD57 using C. pseudotuberculosis strain 1002 as reference, wherein small genomic insertions and deletions were identified. The comparative analysis of the genome sequence provides means to better understand the host pathogen interactions of this strain and can also help us to understand the molecular and genetic basis of virulence of this bacterium.

Complete genome sequence of Streptococcus agalactiae strain GBS85147 serotype of type Ia isolated from human oropharynx.

Streptococcus agalactiae, also referred to as Group B Streptococcus, is a frequent resident of the rectovaginal tract in humans, and a major cause of neonatal infection. The pathogen can also infect adults with underlying disease, particularly the elderly and immunocompromised ones. In addition, S. agalactiae is a known fish pathogen, which compromises food safety and represents a zoonotic hazard. This study provides valuable structural, functional and evolutionary genomic information of a human S. agalactiae serotype Ia (ST-103) GBS85147 strain isolated from the oropharynx of an adult patient from Rio de Janeiro, thereby representing the first human isolate in Brazil. We used the Ion Torrent PGM platform with the 200 bp fragment library sequencing kit. The sequencing generated 578,082,183 bp, distributed among 2,973,022 reads, resulting in an approximately 246-fold mean coverage depth and was assembled using the Mira Assembler v3.9.18. The S. agalactiae strain GBS85147 comprises of a circular chromosome with a final genome length of 1,996,151 bp containing 1,915 protein-coding genes, 18 rRNA, 63 tRNA, 2 pseudogenes and a G + C content of 35.48 %.

Genome Sequence of Corynebacterium ulcerans Strain FRC11.

Here, we present the genome sequence of Corynebacterium ulcerans strain FRC11. The genome includes one circular chromosome of 2,442,826 bp (53.35% G+C content), and 2,210 genes were predicted, 2,146 of which are putative protein-coding genes, with 12 rRNAs and 51 tRNAs; 1 pseudogene was also identified.

Genome Sequence of Corynebacterium ulcerans Strain 210932.

In this work, we present the complete genome sequence of Corynebacterium ulcerans strain 210932, isolated from a human. The species is an emergent pathogen that infects a variety of wild and domesticated animals and humans. It is associated with a growing number of cases of a diphtheria-like disease around the world.