Here we report the complete genome sequence of Streptococcus agalactiae strain 874391. This serotype III isolate is a member of the hypervirulent sequence type 17 (ST-17) lineage that causes a disproportionate number of cases of invasive disease in humans and mammals. A brief historical context of the strain is discussed. Copyright © 2017 Sullivan et al.
Streptococcus pneumoniae (pneumococcus) displays broad tissue tropism and infects multiple body sites in the human host. However, infections of the conjunctiva are limited to strains within a distinct phyletic group with multilocus sequence types ST448, ST344, ST1186, ST1270, and ST2315. In this study, we sequenced the genomes of six pneumococcal strains isolated from eye infections. The conjunctivitis isolates are grouped in a distinct phyletic group together with a subset of nasopharyngeal isolates. The keratitis (infection of the cornea) and endophthalmitis (infection of the vitreous body) isolates are grouped with the remainder of pneumococcal strains. Phenotypic characterization is consistent with morphological differences associated with the distinct phyletic group. Specifically, isolates from the distinct phyletic group form aggregates in planktonic cultures and chain-like structures in biofilms grown on abiotic surfaces. To begin to investigate the association between genotype and epidemiology, we focused on a predicted surface-exposed adhesin (SspB) encoded exclusively by this distinct phyletic group. Phylogenetic analysis of the gene encoding SspB in the context of a streptococcal species tree suggests that sspB was acquired by lateral gene transfer from Streptococcus suis. Furthermore, an sspB deletion mutant displays decreased adherence to cultured cells from the ocular epithelium compared to the isogenic wild-type and complemented strains. Together these findings suggest that acquisition of genes from outside the species has contributed to pneumococcal tissue tropism by enhancing the ability of a subset of strains to infect the ocular epithelium causing conjunctivitis. IMPORTANCE Changes in the gene content of pathogens can modify their ability to colonize and/or survive in different body sites in the human host. In this study, we investigate a gene acquisition event and its role in the pathogenesis of Streptococccus pneumoniae (pneumococcus). Our findings suggest that the gene encoding the predicted surface protein SspB has been transferred from Streptococcus suis (a distantly related streptococcal species) into a distinct set of pneumococcal strains. This group of strains distinguishes itself from the remainder of pneumococcal strains by extensive differences in genomic composition and by the ability to cause conjunctivitis. We find that the presence of sspB increases adherence of pneumococcus to the ocular epithelium. Thus, our data support the hypothesis that a subset of pneumococcal strains has gained genes from neighboring species that enhance their ability to colonize the epithelium of the eye, thus expanding into a new niche.
Campylobacter avium is a thermotolerant Campylobacter species that has been isolated from poultry. C. avium was also the second hippuricase-positive species to be identified within Campylobacter Here, we present the genome sequence of the C. avium type strain LMG 24591 (=CCUG 56292(T)), isolated in 2006 from a broiler chicken in Italy. Copyright © 2017 Miller et al.
Listeria monocytogenes is one of the most heat-resistant non-spore-forming food-borne pathogens and poses a notable risk to food safety, particularly when mild heat treatments are used in food processing and preparation. While general heat stress properties and response mechanisms of L. monocytogenes have been described, accessory mechanisms providing particular L. monocytogenes strains with the advantage of enhanced heat resistance are unknown. Here, we report plasmid-mediated heat resistance of L. monocytogenes for the first time. This resistance is mediated by the ATP-dependent protease ClpL. We tested the survival of two wild-type L. monocytogenes strains-both of serotype 1/2c, sequence type ST9, and high sequence identity-at high temperatures and compared their genome composition in order to identify genetic mechanisms involved in their heat survival phenotype. L. monocytogenes AT3E was more heat resistant (0.0 CFU/ml log10 reduction) than strain AL4E (1.4 CFU/ml log10 reduction) after heating at 55°C for 40 min. A prominent difference in the genome compositions of the two strains was a 58-kb plasmid (pLM58) harbored by the heat-resistant AT3E strain, suggesting plasmid-mediated heat resistance. Indeed, plasmid curing resulted in significantly decreased heat resistance (1.1 CFU/ml log10 reduction) at 55°C. pLM58 harbored a 2,115-bp open reading frame annotated as an ATP-dependent protease (ClpL)-encoding clpL gene. Introducing the clpL gene into a natively heat-sensitive L. monocytogenes strain (1.2 CFU/ml log10 reduction) significantly increased the heat resistance of the recipient strain (0.4 CFU/ml log10 reduction) at 55°C. Plasmid-borne ClpL is thus a potential predictor of elevated heat resistance in L. monocytogenes. IMPORTANCEListeria monocytogenes is a dangerous food pathogen causing the severe illness listeriosis that has a high mortality rate in immunocompromised individuals. Although destroyed by pasteurization, L. monocytogenes is among the most heat-resistant non-spore-forming bacteria. This poses a risk to food safety, as listeriosis is commonly associated with ready-to-eat foods that are consumed without thorough heating. However, L. monocytogenes strains differ in their ability to survive high temperatures, and comprehensive understanding of the genetic mechanisms underlying these differences is still limited. Whole-genome-sequence analysis and phenotypic characterization allowed us to identify a novel plasmid, designated pLM58, and a plasmid-borne ATP-dependent protease (ClpL), which mediated heat resistance in L. monocytogenes. As the first report on plasmid-mediated heat resistance in L. monocytogenes, our study sheds light on the accessory genetic mechanisms rendering certain L. monocytogenes strains particularly capable of surviving high temperatures-with plasmid-borne ClpL being a potential predictor of elevated heat resistance.
The genus Malassezia includes yeasts that are commonly found on the skin or hair of animals and humans as commensals and are associated with a number of skin disorders. We have previously developed an Agrobacterium tumefaciens transformation system effective for both targeted gene deletion and insertional mutagenesis in Malassezia furfur and M. sympodialis In the present study, these molecular resources were applied to characterize the immunophilin FKBP12 as the target of tacrolimus (FK506), ascomycin, and pimecrolimus, which are calcineurin inhibitors that are used as alternatives to corticosteroids in the treatment of inflammatory skin disorders such as those associated with Malassezia species. While M. furfur and M. sympodialis showed in vitro sensitivity to these agents, fkb1? mutants displayed full resistance to all three of them, confirming that FKBP12 is the target of these calcineurin inhibitors and is essential for their activity. We found that calcineurin inhibitors act additively with fluconazole through an FKBP12-dependent mechanism. Spontaneous M. sympodialis isolates resistant to calcineurin inhibitors had mutations in the gene encoding FKBP12 in regions predicted to affect the interactions between FKBP12 and FK506 based on structural modeling. Due to the presence of homopolymer nucleotide repeats in the gene encoding FKBP12, an msh2? hypermutator of M. sympodialis was engineered and exhibited an increase of more than 20-fold in the rate of emergence of resistance to FK506 compared to that of the wild-type strain, with the majority of the mutations found in these repeats.IMPORTANCEMalassezia species are the most abundant fungal components of the mammalian and human skin microbiome. Although they belong to the natural skin commensal flora of humans, they are also associated with a variety of clinical skin disorders. The standard treatment for Malassezia-associated inflammatory skin infections is topical corticosteroids, although their use has adverse side effects and is not recommended for long treatment periods. Calcineurin inhibitors have been proposed as a suitable alternative to treat patients affected by skin lesions caused by Malassezia Although calcineurin inhibitors are well-known as immunosuppressive drugs, they are also characterized by potent antimicrobial activity. In the present study, we investigated the mechanism of action of FK506 (tacrolimus), ascomycin (FK520), and pimecrolimus in M. furfur and M. sympodialis and found that the conserved immunophilin FKBP12 is the target of these drugs with which it forms a complex that directly binds calcineurin and inhibits its signaling activity. We found that FKBP12 is also required for the additive activity of calcineurin inhibitors with fluconazole. Furthermore, the increasing natural occurrence in fungal pathogen populations of mutator strains poses a high risk for the rapid emergence of drug resistance and adaptation to host defense. This led us to generate an engineered hypermutator msh2? mutant strain of M. sympodialis and genetically evaluate mutational events resulting in a substantially increased rate of resistance to FK506 compared to that of the wild type. Our study paves the way for the novel clinical use of calcineurin inhibitors with lower immunosuppressive activity that could be used clinically to treat a broad range of fungal infections, including skin disorders caused by Malassezia. Copyright © 2017 Ianiri et al.
Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant. Copyright © 2017 Goh et al.
Mycobacterium lepraemurium is the causative agent of murine leprosy, a chronic, granulomatous disease similar to human leprosy. Due to the similar clinical manifestations of human and murine leprosy and the difficulty of growing both bacilli axenically, Mycobacterium leprae and M. lepraemurium were once thought to be closely related, although it was later suggested that M. lepraemurium might be related to Mycobacterium avium In this study, the complete genome of M. lepraemurium was sequenced using a combination of PacBio and Illumina sequencing. Phylogenomic analyses confirmed that M. lepraemurium is a distinct species within the M. avium complex (MAC). The M. lepraemurium genome is 4.05 Mb in length, which is considerably smaller than other MAC genomes, and it comprises 2,682 functional genes and 1,139 pseudogenes, which indicates that M. lepraemurium has undergone genome reduction. An error-prone repair homologue of the DNA polymerase III a-subunit was found to be nonfunctional in M. lepraemurium, which might contribute to pseudogene formation due to the accumulation of mutations in nonessential genes. M. lepraemurium has retained the functionality of several genes thought to influence virulence among members of the MAC.IMPORTANCEMycobacterium lepraemurium seems to be evolving toward a minimal set of genes required for an obligatory intracellular lifestyle within its host, a niche seldom adopted by most mycobacteria, as they are free-living. M. lepraemurium could be used as a model to elucidate functions of genes shared with other members of the MAC. Its reduced gene set can be exploited for studying the essentiality of genes in related pathogenic species, which might lead to discovery of common virulence factors or clarify host-pathogen interactions. M. lepraemurium can be cultivated in vitro only under specific conditions and even then with difficulty. Elucidating the metabolic (in)capabilities of M. lepraemurium will help develop suitable axenic media and facilitate genetic studies. Copyright © 2017 Benjak et al.
In our previous study, Citrobacter werkmanii BF-6 was isolated from an industrial spoilage sample and demonstrated an excellent ability to form biofilms, which could be affected by various environmental factors. However, the genome sequence of this organism has not been reported so far.We report the complete genome sequence of C. werkmanii BF-6 together with the description of the genome features and its annotation. The size of the complete chromosome is 4,929,789 bp with an average coverage of 137×. The chromosome exhibits an average G + C content of 52.0%, and encodes 4570 protein coding genes, 84 tRNA genes, 25 rRNA operons, 3 microsatellite sequences and 34 minisatellite sequences. A previously unknown circular plasmid designated as pCW001 was also found with a length of 212,549 bp and a G + C content of 48.2%. 73.5%, 75.6% and 92.6% of the protein coding genes could be assigned to GO Ontology, KEGG Pathway, and COG (Clusters of Orthologous Groups) categories respectively. C. werkmanii BF-6 and C. werkmanii NRBC 105721 exhibited the closest evolutionary relationships based on 16S ribosomal RNA and core-pan genome assay. Furthermore, C. werkmanii BF-6 exhibits typical bacterial biofilm formation and development. In the RT-PCR experiments, we found that a great number of biofilm related genes, such as bsmA, bssR, bssS, hmsP, tabA, csgA, csgB, csgC, csgD, csgE, and csgG, were involved in C. werkmanii BF-6 biofilm formation.This is the first complete genome of C. werkmanii. Our work highlights the potential genetic mechanisms involved in biofilm formation and paves a way for further application of C. werkmanii in biofilms research.
Babesia ovata, belonging to the phylum Apicomplexa, is an infectious parasite of bovids. It is not associated with the manifestation of severe symptoms, in contrast to other types of bovine babesiosis caused by B. bovis and B. bigemina; however, upon co-infection with Theileria orientalis, it occasionally induces exacerbated symptoms. Asymptomatic chronic infection in bovines is usually observed only for B. ovata. Comparative genomic analysis could potentially reveal factors involved in these distinguishing characteristics; however, the genomic and molecular basis of these phenotypes remains elusive, especially in B. ovata. From a technical perspective, the current development of a very long read sequencer, MinION, will facilitate the obtainment of highly integrated genome sequences. Therefore, we applied next-generation sequencing to acquire a high-quality genome of the parasite, which provides fundamental information for understanding apicomplexans.The genome was assembled into 14,453,397 bp in size with 5031 protein-coding sequences (91 contigs and N50 = 2,090,503 bp). Gene family analysis revealed that ves1 alpha and beta, which belong to multigene families in B. bovis, were absent from B. ovata, the same as in B. bigemina. Instead, ves1a and ves1b, which were originally specified in B. bigemina, were present. The B. ovata and B. bigemina ves1a configure one cluster together even though they divided into two sub-clusters according to the spp. In contrast, the ves1b cluster was more dispersed and the overlap among B. ovata and B. bigemina was limited. The observed redundancy and rapid evolution in sequence might reflect the adaptive history of these parasites. Moreover, same candidate genes which potentially involved in the distinct phenotypes were specified by functional analysis. An anamorsin homolog is one of them. The human anamorsin is involved in hematopoiesis and the homolog was present in B. ovata but absent in B. bigemina which causes severe anemia.Taking these findings together, the differences demonstrated by comparative genomics potentially explain the evolutionary history of these parasites and the differences in their phenotypes. Besides, the draft genome provides fundamental information for further characterization and understanding of these parasites.
The ST313 sequence type of Salmonella Typhimurium causes invasive non-typhoidal salmonellosis and was thought to be confined to sub-Saharan Africa. Two distinct phylogenetic lineages of African ST313 have been identified.We analysed the whole genome sequences of S. Typhimurium isolates from UK patients that were generated following the introduction of routine whole-genome sequencing (WGS) of Salmonella enterica by Public Health England in 2014.We found that 2.7% (84/3147) of S. Typhimurium from patients in England and Wales were ST313 and were associated with gastrointestinal infection. Phylogenetic analysis revealed novel diversity of ST313 that distinguished UK-linked gastrointestinal isolates from African-associated extra-intestinal isolates. The majority of genome degradation of African ST313 lineage 2 was conserved in the UK-ST313, but the African lineages carried a characteristic prophage and antibiotic resistance gene repertoire. These findings suggest that a strong selection pressure exists for certain horizontally acquired genetic elements in the African setting. One UK-isolated lineage 2 strain that probably originated in Kenya carried a chromosomally located bla CTX-M-15, demonstrating the continual evolution of this sequence type in Africa in response to widespread antibiotic usage.The discovery of ST313 isolates responsible for gastroenteritis in the UK reveals new diversity in this important sequence type. This study highlights the power of routine WGS by public health agencies to make epidemiologically significant deductions that would be missed by conventional microbiological methods. We speculate that the niche specialisation of sub-Saharan African ST313 lineages is driven in part by the acquisition of accessory genome elements.
Babesia bovis, is a tick borne apicomplexan parasite responsible for important cattle losses globally. Babesia parasites have a complex life cycle including asexual replication in the mammalian host and sexual reproduction in the tick vector. Novel control strategies aimed at limiting transmission of the parasite are needed, but transmission blocking vaccine candidates remain undefined. Expression of HAP2 has been recognized as critical for the fertilization of parasites in the Babesia-related Plasmodium, and is a leading candidate for a transmission blocking vaccine against malaria. Hereby we identified the B. bovis hap2 gene and demonstrated that it is widely conserved and differentially transcribed during development within the tick midgut, but not by blood stage parasites. The hap2 gene was disrupted by transfecting B. bovis with a plasmid containing the flanking regions of the hap2 gene and the GPF-BSD gene under the control of the ef-1a-B promoter. Comparison of in vitro growth between a hap2-KO B. bovis clonal line and its parental wild type strain showed that HAP2 is not required for the development of B. bovis in erythrocytes. However, xanthurenic acid-in vitro induction experiments of sexual stages of parasites recovered after tick transmission resulted in surface expression of HAP2 exclusively in sexual stage induced parasites. In addition, hap2-KO parasites were not able to develop such sexual stages as defined both by morphology and by expression of the B. bovis sexual marker genes 6-Cys A and B. Together, the data strongly suggests that tick midgut stage differential expression of hap2 is associated with the development of B. bovis sexual forms. Overall these studies are consistent with a role of HAP2 in tick stages of the parasite and suggest that HAP2 is a potential candidate for a transmission blocking vaccine against bovine babesiosis.
Armillaria species are both devastating forest pathogens and some of the largest terrestrial organisms on Earth. They forage for hosts and achieve immense colony sizes via rhizomorphs, root-like multicellular structures of clonal dispersal. Here, we sequenced and analysed the genomes of four Armillaria species and performed RNA sequencing and quantitative proteomic analysis on the invasive and reproductive developmental stages of A.?ostoyae. Comparison with 22 related fungi revealed a significant genome expansion in Armillaria, affecting several pathogenicity-related genes, lignocellulose-degrading enzymes and lineage-specific genes expressed during rhizomorph development. Rhizomorphs express an evolutionarily young transcriptome that shares features with the transcriptomes of both fruiting bodies and vegetative mycelia. Several genes show concomitant upregulation in rhizomorphs and fruiting bodies and share cis-regulatory signatures in their promoters, providing genetic and regulatory insights into complex multicellularity in fungi. Our results suggest that the evolution of the unique dispersal and pathogenicity mechanisms of Armillaria might have drawn upon ancestral genetic toolkits for wood-decay, morphogenesis and complex multicellularity.
Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.
The purpose of this study was to determine the plasmid architecture and context of resistance genes in multi-drug resistant (MDR) Escherichia coli strains isolated from urinary tract infections in dogs. Illumina and single-molecule real-time (SMRT) sequencing were applied to assemble the complete genomes of E. coli strains associated with clinical urinary tract infections, which were either phenotypically MDR or drug susceptible. This revealed that multiple distinct families of plasmids were associated with building an MDR phenotype. Plasmid-mediated AmpC (CMY-2) beta-lactamase resistance was associated with a clonal group of IncI1 plasmids that has remained stable in isolates collected up to a decade apart. Other plasmids, in particular those with an IncF replicon type, contained other resistance gene markers, so that the emergence of these MDR strains was driven by the accumulation of multiple plasmids, up to 5 replicons in specific cases. This study indicates that vulnerable patients, often with complex clinical histories provide a setting leading to the emergence of MDR E. coli strains in clonally distinct commensal backgrounds. While it is known that horizontally-transferred resistance supplements uropathogenic strains of E. coli such as ST131, our study demonstrates that the selection of an MDR phenotype in commensal E. coli strains can result in opportunistic infections in vulnerable patient populations. These strains provide a reservoir for the onward transfer of resistance alleles into more typically pathogenic strains and provide opportunities for the coalition of resistance and virulence determinants on plasmids as evidenced by the IncF replicons characterised in this study. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
Pasteurella multocida causes pneumonic pasteurellosis and hemorrhagic septicemia (HS) in large ruminants. In this study, we determined the complete genome sequence of P. multocida strain PMTB2.1 capsular serotype A isolated from buffaloes that died of septicemia. Copyright © 2017 Jabeen et al.
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