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September 22, 2019

Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance.

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer’s and Huntington’s disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens.IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, “On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you.” Copyright © 2018 Bohlmann et al.

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September 22, 2019

Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains.

The recent discovery of the plasmid-mediated mcr-1 gene conferring resistance to colistin is of clinical concern. The worldwide screening of this resistance mechanism among samples of different origins has highlighted the urgent need to improve the detection of colistin-resistant isolates in clinical microbiology laboratories. Currently, phenotypic methods used to detect colistin resistance are not necessarily suitable as the main characteristic of the mcr genes is the low level of resistance that they confer, close to the clinical breakpoint recommended jointly by the CLSI and EUCAST expert systems (S?=?2?mg/L and R?>?2?mg/L). In this context, susceptibility testing recommendations for polymyxins have evolved and are becoming difficult to implement in routine laboratory work. The large number of mechanisms and genes involved in colistin resistance limits the access to rapid detection by molecular biology. It is therefore necessary to implement well-defined protocols using specific tools to detect all colistin-resistant bacteria. This review aims to summarize the current clinical microbiology diagnosis techniques and their ability to detect all colistin resistance mechanisms and describe new tools specifically developed to assess plasmid-mediated colistin resistance. Phenotyping, susceptibility testing, and genotyping methods are presented, including an update on recent studies related to the development of specific techniques.

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September 22, 2019

De novo assembly of the Pasteuria penetrans genome reveals high plasticity, host dependency, and BclA-like collagens.

Pasteuria penetrans is a gram-positive endospore forming bacterial parasite of Meloidogyne spp. the most economically damaging genus of plant parasitic nematodes globally. The obligate antagonistic nature of P. penetrans makes it an attractive candidate biological control agent. However, deployment of P. penetrans for this purpose is inhibited by a lack of understanding of its metabolism and the molecular mechanics underpinning parasitism of the host, in particular the initial attachment of the endospore to the nematode cuticle. Several attempts to assemble the genomes of species within this genus have been unsuccessful. Primarily this is due to the obligate parasitic nature of the bacterium which makes obtaining genomic DNA of sufficient quantity and quality which is free from contamination challenging. Taking advantage of recent developments in whole genome amplification, long read sequencing platforms, and assembly algorithms, we have developed a protocol to generate large quantities of high molecular weight genomic DNA from a small number of purified endospores. We demonstrate this method via genomic assembly of P. penetrans. This assembly reveals a reduced genome of 2.64Mbp estimated to represent 86% of the complete sequence; its reduced metabolism reflects widespread reliance on the host and possibly associated organisms. Additionally, apparent expansion of transposases and prediction of partial competence pathways suggest a high degree of genomic plasticity. Phylogenetic analysis places our sequence within the Bacilli, and most closely related to Thermoactinomyces species. Seventeen predicted BclA-like proteins are identified which may be involved in the determination of attachment specificity. This resource may be used to develop in vitro culture methods and to investigate the genetic and molecular basis of attachment specificity.

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September 22, 2019

Mutators as drivers of adaptation in Streptococcus and a risk factor for host jumps and vaccine escape

Heritable hypermutable strains deficient in DNA repair genes (mutators) facilitate microbial adaptation as they may rapidly generate beneficial mutations. Mutators deficient in mismatch (MMR) and oxidised guanine (OG) repair are abundant in clinical samples and show increased adaptive potential in experimental infection models but their role in pathoadaptation is poorly understood. Here we investigate the role of mutators in epidemiology and evolution of the broad host pathogen, Streptococcus iniae, employing 80 strains isolated globally over 40 years. We determine phylogenetic relationship among S. iniae using 10,267 non-recombinant core genome single nucleotide polymorphisms (SNPs), estimate their mutation rate by fluctuation analysis, and detect variation in major MMR (mutS, mutL, dnaN, recD2, rnhC) and OG (mutY, mutM, mutX) genes. S. iniae mutation rate phenotype and genotype are strongly associated with phylogenetic diversification and variation in major streptococcal virulence determinants (capsular polysaccharide, hemolysin, cell chain length, resistance to oxidation, and biofilm formation). Furthermore, profound changes in virulence determinants observed in mammalian isolates (atypical host) and vaccine-escape isolates found in bone (atypical tissue) of vaccinated barramundi are linked to multiple MMR and OG variants and unique mutation rates. This implies that adaptation to new host taxa, new host tissue, and to immunity of a vaccinated host is promoted by mutator strains. Our findings support the importance of mutation rate dynamics in evolution of pathogenic bacteria, in particular adaptation to a drastically different immunological setting that occurs during host jump and vaccine escape events.Importance Host immune response is a powerful selective pressure that drives diversification of pathogenic microorganisms and, ultimately, evolution of new strains. Major adaptive events in pathogen evolution, such as transmission to a new host species or infection of vaccinated hosts, require adaptation to a drastically different immune landscape. Such adaptation may be favoured by hypermutable strains (or mutators) that are defective in normal DNA repair and consequently capable of generating multiple potentially beneficial and compensatory mutations. This permits rapid adjustment of virulence and antigenicity in a new immunological setting. Here we show that mutators, through mutations in DNA repair genes and corresponding shifts in mutation rate, are associated with major diversification events and virulence evolution in the broad host-range pathogen Streptococcus iniae. We show that mutators underpin infection of vaccinated hosts, transmission to new host species and the evolution of new strains.

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September 21, 2019

Functional analysis of the first complete genome sequence of a multidrug resistant sequence type 2 Staphylococcus epidermidis.

Staphylococcus epidermidis is a significant opportunistic pathogen of humans. The ST2 lineage is frequently multidrug resistant and accounts for most of the clinical disease worldwide. However, there are no publically available, closed ST2 genomes and pathogenesis studies have not focused on these strains. We report the complete genome and methylome of BPH0662, a multidrug resistant, hospital adapted, ST2 S. epidermidis, and describe the correlation between resistome and phenotype, as well as demonstrate its relationship to publically available, international ST2 isolates. Furthermore, we delineate the methylome determined by the two type I restriction modification systems present in BPH0662 through heterologous expression in Escherichia coli, allowing the assignment of each system to its corresponding target recognition motif. As the first complete ST2 S. epidermidis genome, BPH0662 provides a valuable reference for future genomic studies of this clinically relevant lineage. Defining the methylome and the construction of these E. coli hosts provides the foundation for the development of molecular tools to bypass restriction modification systems in this lineage that has hitherto proven intractable.

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September 21, 2019

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7.

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.

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September 21, 2019

Characterization of multi-drug resistant Enterococcus faecalis isolated from cephalic recording chambers in research macaques (Macaca spp.).

Nonhuman primates are commonly used for cognitive neuroscience research and often surgically implanted with cephalic recording chambers for electrophysiological recording. Aerobic bacterial cultures from 25 macaques identified 72 bacterial isolates, including 15 Enterococcus faecalis isolates. The E. faecalis isolates displayed multi-drug resistant phenotypes, with resistance to ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, bacitracin, and erythromycin, as well as high-level aminoglycoside resistance. Multi-locus sequence typing showed that most belonged to two E. faecalis sequence types (ST): ST 4 and ST 55. The genomes of three representative isolates were sequenced to identify genes encoding antimicrobial resistances and other traits. Antimicrobial resistance genes identified included aac(6′)-aph(2″), aph(3′)-III, str, ant(6)-Ia, tetM, tetS, tetL, ermB, bcrABR, cat, and dfrG, and polymorphisms in parC (S80I) and gyrA (S83I) were observed. These isolates also harbored virulence factors including the cytolysin toxin genes in ST 4 isolates, as well as multiple biofilm-associated genes (esp, agg, ace, SrtA, gelE, ebpABC), hyaluronidases (hylA, hylB), and other survival genes (ElrA, tpx). Crystal violet biofilm assays confirmed that ST 4 isolates produced more biofilm than ST 55 isolates. The abundance of antimicrobial resistance and virulence factor genes in the ST 4 isolates likely relates to the loss of CRISPR-cas. This macaque colony represents a unique model for studying E. faecalis infection associated with indwelling devices, and provides an opportunity to understand the basis of persistence of this pathogen in a healthcare setting.

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September 21, 2019

Whole genome sequence of the soybean aphid, Aphis glycines.

Aphids are emerging as model organisms for both basic and applied research. Of the 5,000 estimated species, only three aphids have published whole genome sequences: the pea aphid Acyrthosiphon pisum, the Russian wheat aphid, Diuraphis noxia, and the green peach aphid, Myzus persicae. We present the whole genome sequence of a fourth aphid, the soybean aphid (Aphis glycines), which is an extreme specialist and an important invasive pest of soybean (Glycine max). The availability of genomic resources is important to establish effective and sustainable pest control, as well as to expand our understanding of aphid evolution. We generated a 302.9 Mbp draft genome assembly for Ap. glycines using a hybrid sequencing approach. This assembly shows high completeness with 19,182 predicted genes, 92% of known Ap. glycines transcripts mapping to contigs, and substantial continuity with a scaffold N50 of 174,505 bp. The assembly represents 95.5% of the predicted genome size of 317.1 Mbp based on flow cytometry. Ap. glycines contains the smallest known aphid genome to date, based on updated genome sizes for 19 aphid species. The repetitive DNA content of the Ap. glycines genome assembly (81.6 Mbp or 26.94% of the 302.9 Mbp assembly) shows a reduction in the number of classified transposable elements compared to Ac. pisum, and likely contributes to the small estimated genome size. We include comparative analyses of gene families related to host-specificity (cytochrome P450’s and effectors), which may be important in Ap. glycines evolution. This Ap. glycines draft genome sequence will provide a resource for the study of aphid genome evolution, their interaction with host plants, and candidate genes for novel insect control methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

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September 21, 2019

Decreased fitness and virulence in ST10 Escherichia coli harboring blaNDM-5 and mcr-1 against a ST4981 strain with blaNDM-5.

Although coexistence of blaNDM-5 and mcr-1 in Escherichia coli has been reported, little is known about the fitness and virulence of such strains. Three carbapenem-resistant Escherichia coli (GZ1, GZ2, and GZ3) successively isolated from one patient in 2015 were investigated for microbiological fitness and virulence. GZ1 and GZ2 were also resistant to colistin. To verify the association between plasmids and fitness, growth kinetics of the transconjugants were performed. We also analyzed genomic sequences of GZ2 and GZ3 using PacBio sequencing. GZ1 and GZ2 (ST10) co-harbored blaNDM-5 and mcr-1, while GZ3 (ST4981) carried only blaNDM-5. GZ3 demonstrated significantly more rapid growth (P < 0.001) and overgrew GZ2 with a competitive index of 1.0157 (4 h) and 2.5207 (24 h). Increased resistance to serum killing and mice mortality was also identified in GZ3. While GZ2 had four plasmids (IncI2, IncX3, IncHI2, IncFII), GZ3 possessed one plasmid (IncFII). The genetic contexts of blaNDM-5 in GZ2 and GZ3 were identical but inserted into different backbones, IncX3 (102,512 bp) and IncFII (91,451 bp), respectively. The growth was not statistically different between the transconjugants with mcr-1 or blaNDM-5 plasmid and recipient (P = 0.6238). Whole genome sequence analysis revealed that 28 virulence genes were specific to GZ3, potentially contributing to increased virulence of GZ3. Decreased fitness and virulence in a mcr-1 and blaNDM-5 co-harboring ST10 E. coli was found alongside a ST4981 strain with only blaNDM-5. Acquisition of mcr-1 or blaNDM-5 plasmid did not lead to considerable fitness costs, indicating the potential for dissemination of mcr-1 and blaNDM-5 in Enterobacteriaceae.

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September 21, 2019

A distinct and genetically diverse lineage of the hybrid fungal pathogen Verticillium longisporum population causes stem striping in British oilseed rape.

Population genetic structures illustrate evolutionary trajectories of organisms adapting to differential environmental conditions. Verticillium stem striping disease on oilseed rape was mainly observed in continental Europe, but has recently emerged in the United Kingdom. The disease is caused by the hybrid fungal species Verticillium longisporum that originates from at least three separate hybridization events, yet hybrids between Verticillium progenitor species A1 and D1 are mainly responsible for Verticillium stem striping. We reveal a hitherto un-described dichotomy within V. longisporum lineage A1/D1 that correlates with the geographic distribution of the isolates with an ‘A1/D1 West’ and an ‘A1/D1 East’ cluster. Genome comparison between representatives of the A1/D1 West and East clusters excluded population distinctiveness through separate hybridization events. Remarkably, the A1/D1 West population that is genetically more diverse than the entire A1/D1 East cluster caused the sudden emergence of Verticillium stem striping in the UK, whereas in continental Europe Verticillium stem striping is predominantly caused by the more genetically uniform A1/D1 East population. The observed genetic diversity of the A1/D1 West population argues against a recent introduction of the pathogen into the UK, but rather suggests that the pathogen previously established in the UK and remained latent or unnoticed as oilseed rape pathogen until recently.© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 21, 2019

PacBio assembly of a Plasmodium knowlesi genome sequence with Hi-C correction and manual annotation of the SICAvar gene family.

Plasmodium knowlesi has risen in importance as a zoonotic parasite that has been causing regular episodes of malaria throughout South East Asia. The P. knowlesi genome sequence generated in 2008 highlighted and confirmed many similarities and differences in Plasmodium species, including a global view of several multigene families, such as the large SICAvar multigene family encoding the variant antigens known as the schizont-infected cell agglutination proteins. However, repetitive DNA sequences are the bane of any genome project, and this and other Plasmodium genome projects have not been immune to the gaps, rearrangements and other pitfalls created by these genomic features. Today, long-read PacBio and chromatin conformation technologies are overcoming such obstacles. Here, based on the use of these technologies, we present a highly refined de novo P. knowlesi genome sequence of the Pk1(A+) clone. This sequence and annotation, referred to as the ‘MaHPIC Pk genome sequence’, includes manual annotation of the SICAvar gene family with 136 full-length members categorized as type I or II. This sequence provides a framework that will permit a better understanding of the SICAvar repertoire, selective pressures acting on this gene family and mechanisms of antigenic variation in this species and other pathogens.

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September 21, 2019

The kinetoplastid-infecting Bodo saltans virus (BsV), a window into the most abundant giant viruses in the sea.

Giant viruses are ecologically important players in aquatic ecosystems that have challenged concepts of what constitutes a virus. Herein, we present the giant Bodo saltans virus (BsV), the first characterized representative of the most abundant group of giant viruses in ocean metagenomes, and the first isolate of a klosneuvirus, a subgroup of the Mimiviridae proposed from metagenomic data. BsV infects an ecologically important microzooplankton, the kinetoplastid Bodo saltans. Its 1.39 Mb genome encodes 1227 predicted ORFs, including a complex replication machinery. Yet, much of its translational apparatus has been lost, including all tRNAs. Essential genes are invaded by homing endonuclease-encoding self-splicing introns that may defend against competing viruses. Putative anti-host factors show extensive gene duplication via a genomic accordion indicating an ongoing evolutionary arms race and highlighting the rapid evolution and genomic plasticity that has led to genome gigantism and the enigma that is giant viruses.© 2018, Deeg et al.

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September 21, 2019

Chromulinavorax destructans, a pathogenic TM6 bacterium with an unusual replication strategy targeting protist mitochondrion

Most of the diversity of microbial life is not available in culture, and as such we lack even a fundamental understanding of the biological diversity of several branches on the tree of life. One branch that is highly underrepresented is the candidate phylum TM6, also known as the Dependentiae. Their biology is known only from reduced genomes recovered from metagenomes around the world and two isolates infecting amoebae, all suggest that they live highly host-associated lifestyles as parasites or symbionts. Chromulinavorax destructans is an isolate from the TM6/Dependentiae that infects and lyses the abundant heterotrophic flagellate, Spumella elongata. Chromulinavorax destructans is characterized by a high degree of reduction and specialization for infection, so much so it was discovered in a screen for giant viruses. Its 1.2 Mb genome shows no metabolic potential and C. destructans instead relies on extensive transporter system to import nutrients, and even energy in the form of ATP from the host. Accordingly, it replicates in a viral-like fashion, while extensively reorganizing and expanding the host mitochondrion. 44% of proteins contain signal sequences for secretion, which includes many proteins of unknown function as well as 98 copies of ankyrin-repeat domain proteins, known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus.

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September 21, 2019

Multi-Locus Variable number of tandem repeat Analysis (MLVA) of Yersinia ruckeri confirms the existence of host-specificity, geographic endemism and anthropogenic dissemination of virulent clones.

A Multi-Locus Variable number of tandem repeat Analysis (MLVA) assay was developed for epizootiological study of the internationally significant fish pathogen Yersinia ruckeri, which causes yersiniosis in salmonids. The assay involves amplification of ten Variable Number of Tandem Repeat (VNTR) loci in two five-plex PCR reactions, followed by capillary electrophoresis. A collection of 484 Y. ruckeri isolates, originating from various biological sources and collected from four continents over seven decades, was analysed. Minimum spanning tree cluster analysis of MLVA profiles separated the studied population into nine major clonal complexes, and a number of minor clusters and singletons. The major clonal complexes could be associated with host species, geographic origin and serotype. A single large clonal complex of serotype O1 isolates dominating the yersiniosis situation in international rainbow trout farming suggests anthropogenic spread of this clone, possibly related to transport of fish. Moreover, sub-clustering within this clonal complex indicates putative transmission routes and multiple biotype shift events. In contrast to the situation in rainbow trout, Y. ruckeri strains associated with disease in Atlantic salmon appear as more or less geographically isolated clonal complexes. A single complex of serotype O1 exclusive to Norway was found to be responsible for almost all major yersiniosis outbreaks in modern Norwegian salmon farming, and site-specific sub-clustering further indicates persistent colonisation of freshwater farms in Norway. Identification of genetically diverse Y. ruckeri isolates from clinically healthy fish and environmental sources also suggests the widespread existence of less virulent or avirulent strains.Importance This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for Yersinia ruckeri infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable, robust, and provides clear, unambiguous and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any Y. ruckeri strain may rapidly be placed in a global epizootiological context. Copyright © 2018 Gulla et al.

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