Extended-spectrum Beta-lactamase gene sequences in gram-negative saprophytes on retail organic and nonorganic spinach

A substantial proportion of infections caused by drug-resistant Gram-negative bacteria (GNB) in community and health care settings are recognized to be caused by evolutionarily related GNB strains. Their global spread has been suggested to occur due to human activities, such as food trade and travel. These multidrug-resistant GNB pathogens often harbor mobile drug resistance genes that are highly conserved in their sequences. Because they appear across different GNB species, these genes may have origins other than human pathogens. We hypothesized that saprophytes in common human food products may serve as a reservoir for such genes. Between July 2007 and April 2008, we examined 25 batches of prepackaged retail spinach for cultivatable GNB population structure by 16S rRNA gene sequencing and for antimicrobial drug susceptibility testing and the presence of extended-spectrum beta-lactamase (ESBL) genes. We found 20 recognized GNB species among 165 (71%) of 231 randomly selected colonies cultured from spinach. Twelve strains suspected to express ESBLs based on resistance to cefotaxime and ceftazidime were further examined for bla(CTX-M) and bla(TEM) genes. We found a 712-bp sequence in Pseudomonas teessidea that was 100% identical to positions 10 to 722 of an 876-bp bla(CTX-M-15) gene of an E. coli strain. Additionally, we identified newly recognized ESBL bla(RAHN-2) sequences from Rahnella aquatilis. These observations demonstrate that saprophytes in common fresh produce can harbor drug resistance genes that are also found in internationally circulating strains of GNB pathogens; such a source may thus serve as a reservoir for drug resistance genes that ultimately enter pathogens to affect human health.     

A domain-centric analysis of oomycete plant pathogen genomes reveals unique protein organization

Oomycetes comprise a diverse group of organisms that morphologically resemble fungi but belong to the stramenopile lineage within the supergroup of chromalveolates. Recent studies have shown that plant pathogenic oomycetes have expanded gene families that are possibly linked to their pathogenic lifestyle. We analyzed the protein domain organization of 67 eukaryotic species including four oomycete and five fungal plant pathogens. We detected 246 expanded domains in fungal and oomycete plant pathogens. The analysis of genes differentially expressed during infection revealed a significant enrichment of genes encoding expanded domains as well as signal peptides linking a substantial part of these genes to pathogenicity. Overrepresentation and clustering of domain abundance profiles revealed domains that might have important roles in host-pathogen interactions but, as yet, have not been linked to pathogenicity. The number of distinct domain combinations (bigrams) in oomycetes was significantly higher than in fungi. We identified 773 oomycete-specific bigrams, with the majority composed of domains common to eukaryotes. The analyses enabled us to link domain content to biological processes such as host-pathogen interaction, nutrient uptake, or suppression and elicitation of plant immune responses. Taken together, this study represents a comprehensive overview of the domain repertoire of fungal and oomycete plant pathogens and points to novel features like domain expansion and species-specific bigram types that could, at least partially, explain why oomycetes are such remarkable plant pathogens.     

The Mammalian Pathogenic Oomycetes

The oomycetes are fungal-like microbes similar to those found within some members of the kingdom Fungi. Although these two groups of microbes share morphological features, there are several contrasting differences: a) phylogenetic analysis placed the oomycetes basal to plants and green algae; b) oomycetes lack ergosterol in their cytoplasmic membrane; c) chitin is not the main compound in the cell wall of oomycetes; and d) asexual reproduction in the oomycetes occurs by the development of sporangia containing numerous biflagellate zoospores. Pythium insidiosum was considered to be the only oomycete pathogenic for mammals. However, in 1999, Grooters reported that several dogs were diagnosed with an unusual oomycete in the genus Lagenidium causing extensive cutaneous and subcutaneous infections. Thereafter, the infection has been also reported in humans and cats, and it could possibly affect other mammalian species as well. This review highlights the epidemiological, clinical and pathological features, as well as the diagnosis and management of the infections caused by this unique group of mammalian pathogenic oomycetes.     

Mass spectrometry-based proteomics for the detection of plant pathogens

Plant diseases caused by fungi, oomycetes, viruses, and bacteria are devastating both to the economy and to the food supply of a nation. Therefore, the development of new, rapid methods to identify these pathogens is a highly important area of research that is of international concern. MS-based proteomics has become a powerful and increasingly popular approach to not only identify these pathogens, but also to better understand their biology. However, there is a distinction between identifying a pathogen protein and identifying a pathogen based upon the detection of one of its proteins and this must be considered before the general application of MS for plant pathogen detection is made. There has been a recent push in the proteomics community to make data from large-scale proteomics experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MS as a universal plant pathogen detection technology.     

Evolution of filamentous plant pathogens: gene exchange across eukaryotic kingdoms

Filamentous fungi and oomycetes are eukaryotic microorganisms that grow by producing networks of thread-like hyphae, which secrete enzymes to break down complex nutrients, such as wood and plant material, and recover the resulting simple sugars and amino acids by osmotrophy. These organisms are extremely similar in both appearance and lifestyle and include some of the most economically important plant pathogens . However, the morphological similarity of fungi and oomycetes is misleading because they represent some of the most distantly related eukaryote evolutionary groupings, and their shared osmotrophic growth habit is interpreted as being the result of convergent evolution . The fungi branch with the animals, whereas the oomycetes branch with photosynthetic algae as part of the Chromalveolata . In this report, we provide strong phylogenetic evidence that multiple horizontal gene transfers (HGT) have occurred from filamentous ascomycete fungi to the distantly related oomycetes. We also present evidence that a subset of the associated gene families was initially the product of prokaryote-to-fungi HGT. The predicted functions of the gene products associated with fungi-to-oomycete HGT suggest that this process has played a significant role in the evolution of the osmotrophic, filamentous lifestyle on two separate branches of the eukaryote tree.     

Biotechnological potential of engineering pathogen effector proteins for use in plant disease management

A key component in the management of many diseases of crops is the use of plant disease resistance genes. However, the discovery and then sequence identification of these plant genes is challenging, whereas the characterization of the molecules that they recognize, the effector/avirulence products in pathogens, is often considerably more straight forward. Effectors are small proteins secreted by pathogens that can play major roles in modulating a plant's defense against attack. Effectors can be used to guide breeding of resistance genes, to trigger defense responses, and are part of integrated disease management strategies for crop protection. This review covers the role of effector-driven biotechnology in controlling plant diseases caused by fungi or oomycetes. Given that multi-billion dollar agriculture crops are based in some cases on plants recognizing just a handful of such effector proteins, there is considerable scope to use more fully effector proteins as a biotechnology resource in agriculture.     

Plasmodesmata: the battleground against intruders

Plasmodesmata are intercellular channels that establish a symplastic communication pathway between neighboring cells in plants. Owing to this role, opportunistic microbial pathogens have evolved to exploit plasmodesmata as gateways to spread infection from cell to cell within the plant. However, although these pathogens have acquired the capacity to breach the plasmodesmal trafficking pathway, plants are unlikely to relinquish control over a structure essential for their survival so easily. In this review, we examine evidence that suggests plasmodesmata play an active role in plant immunity against viral, fungal and bacterial pathogens. We discuss how these pathogens differ in their lifestyles and infection modes, and present the defense strategies that plants have adopted to prevent the intercellular spread of an infection.     

Deciphering microbial landscapes of fish eggs to mitigate emerging diseases

Animals and plants are increasingly suffering from diseases caused by fungi and oomycetes. These emerging pathogens are now recognized as a global threat to biodiversity and food security. Among oomycetes, Saprolegnia species cause significant declines in fish and amphibian populations. Fish eggs have an immature adaptive immune system and depend on nonspecific innate defences to ward off pathogens. Here, meta-taxonomic analyses revealed that Atlantic salmon eggs are home to diverse fungal, oomycete and bacterial communities. Although virulent Saprolegnia isolates were found in all salmon egg samples, a low incidence of Saprolegniosis was strongly correlated with a high richness and abundance of specific commensal Actinobacteria, with the genus Frondihabitans (Microbacteriaceae) effectively inhibiting attachment of Saprolegniato salmon eggs. These results highlight that fundamental insights into microbial landscapes of fish eggs may provide new sustainable means to mitigate emerging diseases.     

Harnessing CRISPR-Cas for oomycete genome editing

Oomycetes are a group of microorganisms that include pathogens responsible for devastating diseases in plants and animals worldwide. Despite their importance, the development of genome editing techniques for oomycetes has progressed more slowly than for model microorganisms. Here, we review recent breakthroughs in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technologies that are expanding the genome editing toolbox for oomycetes – from the original Cas9 study to Cas12a editing, ribonucleoprotein (RNP) delivery, and complementation. We also discuss some of the challenges to applying CRISPR-Cas in oomycetes and potential ways to overcome them. Advances in CRISPR-Cas technologies are being used to illuminate the biology of oomycetes, which ultimately can guide the development of tools for managing oomycete diseases.     

Genome evolution in filamentous plant pathogens: why bigger can be better

Many species of fungi and oomycetes are plant pathogens of great economic importance. Over the past 7 years, the genomes of more than 30 of these filamentous plant pathogens have been sequenced, revealing remarkable diversity in genome size and architecture. Whereas the genomes of many parasites and bacterial symbionts have been reduced over time, the genomes of several lineages of filamentous plant pathogens have been shaped by repeat-driven expansions. In these lineages, the genes encoding proteins involved in host interactions are frequently polymorphic and reside within repeat-rich regions of the genome. Here, we review the properties of these adaptable genome regions and the mechanisms underlying their plasticity, and we illustrate cases in which genome plasticity has contributed to the emergence of new virulence traits. We also discuss how genome expansions may have had an impact on the co-evolutionary conflict between these filamentous plant pathogens and their hosts.     

Drivers for the emergence and re-emergence of vector-borne protozoal and bacterial diseases

In recent years, vector-borne parasitic and bacterial diseases have emerged or re-emerged in many geographical regions causing global health and economic problems that involve humans, livestock, companion animals and wild life. The ecology and epidemiology of vector-borne diseases are affected by the interrelations between three major factors comprising the pathogen, the host (human, animal or vector) and the environment. Important drivers for the emergence and spread of vector-borne parasites include habitat changes, alterations in water storage and irrigation habits, atmospheric and climate changes, immunosuppression by HIV, pollution, development of insecticide and drug resistance, globalization and the significant increase in international trade, tourism and travel. War and civil unrest, and governmental or global management failure are also major contributors to the spread of infectious diseases. The improvement of epidemic understanding and planning together with the development of new diagnostic molecular techniques in the last few decades have allowed researchers to better diagnose and trace pathogens, their origin and routes of infection, and to develop preventive public health and intervention programs. Health care workers, physicians, veterinarians and biosecurity officers should play a key role in future prevention of vector-borne diseases. A coordinated global approach for the prevention of vector-borne diseases should be implemented by international organizations and governmental agencies in collaboration with research institutions.     

Trichoderma/pathogen/plant interaction in pre-harvest food security

Large losses before crop harvesting are caused by plant pathogens, such as viruses, bacteria, oomycetes, fungi, and nematodes. Among these, fungi are the major cause of losses in agriculture worldwide. Plant pathogens are still controlled through application of agrochemicals, causing human disease and impacting environmental and food security. Biological control provides a safe alternative for the control of fungal plant pathogens, because of the ability of biocontrol agents to establish in the ecosystem. Some Trichoderma spp. are considered potential agents in the control of fungal plant diseases. They can interact directly with roots, increasing plant growth, resistance to diseases, and tolerance to abiotic stress. Furthermore, Trichoderma can directly kill fungal plant pathogens by antibiosis, as well as via mycoparasitism strategies. In this review, we will discuss the interactions between Trichoderma/fungal pathogens/plants during the pre-harvest of crops. In addition, we will highlight how these interactions can influence crop production and food security. Finally, we will describe the future of crop production using antimicrobial peptides, plants carrying pathogen-derived resistance, and plantibodies.     

Nep1-like proteins as a target for plant pathogen control

The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.     

Design and development of a DNA array for rapid detection and identification of multiple tomato vascular wilt pathogens

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or Verticillium dahliae, are devastating diseases of tomato (Lycopersicon esculentum) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We show the utility of this array for the sensitive detection of these pathogens from complex substrates like soil, plant tissues and irrigation water, and samples that are collected by tomato growers in their greenhouses.     

Class 1 integrons potentially predating the association with tn402-like transposition genes are present in a sediment microbial community

Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."     

Oomycetes associated with Prunus serotina persist in soil after tree harvest

Soil-borne pathogens can shape forest communities by lowering seedling survivorship. Many soil pathogens can persist long-term as survival spores, but how long pathogens outlive tree hosts in gap soils and whether they continue to affect seedling survival is uncertain. We studied the presence of oomycetes and evaluated seedling performance in soils near live Prunus serotina trees, and 0.5 and 1.5-y-old stumps. We isolated five species of oomycetes from soils, two of which were pathogenic (Pythium intermedium and Pythium irregulare) to Prunus serotina. There was a non-significant ~10.5% increase in conspecific seedling survival in stumps versus live trees, and pathogens were present in soils of all stump ages. The continued presence of pathogens of Prunus serotina in gap soils demonstrates the potential for impacts on conspecific regeneration after tree death, though the slight improvement in survival suggests that these effects may weaken with time.     

The molecular dialog between oomycete effectors and their plant and animal hosts

Oomycetes form a phylogenetically distinct group of eukaryotic microorganisms that include some of the most notorious pathogens of plants and animals. Through the deployment of a remarkably diverse array of effector proteins, oomycete pathogens succeed to overcome host defences and cause infection. Effectors can operate extracellularly or enter living cells where they target diverse subcellular compartments. Genome sequence information indicates that oomycetes express several hundred host-translocating effectors potentially targeting a myriad of host processes. To counteract, plants rely on a wide variety of extra- and intracellular immune receptors facilitating pattern-triggered and effector-triggered immunity, respectively. Similarly, effectors from animal pathogenic oomycetes also target host immune response pathways, which in turn causes the activation of the humoral and adaptive immune system. In this review, we compare plant and animal pathogenic oomycete effectors regarding their type, function, genetic diversity, as well as host responses.     

Modeling antibiotic resistance in hospitals: the impact of minimizing treatment duration

Infections caused by antibiotic-resistant pathogens are a global public health problem. Numerous individual- and population-level factors contribute to the emergence and spread of these pathogens. An individual-based model (IBM), formulated as a system of stochastically determined events, was developed to describe the complexities of the transmission dynamics of antibiotic-resistant bacteria. To simplify the interpretation and application of the model's conclusions, a corresponding deterministic model was created, which describes the average behavior of the IBM over a large number of simulations. The integration of these two model systems provides a quantitative analysis of the emergence and spread of antibiotic-resistant bacteria, and demonstrates that early initiation of treatment and minimization of its duration mitigates antibiotic resistance epidemics in hospitals.     

Recent developments in effector biology of filamentous plant pathogens

Filamentous pathogens, such as plant pathogenic fungi and oomycetes, secrete an arsenal of effector molecules that modulate host innate immunity and enable parasitic infection. It is now well accepted that these effectors are key pathogenicity determinants that enable parasitic infection. In this review, we report on the most interesting features of a representative set of filamentous pathogen effectors and highlight recent findings. We also list and describe all the linear motifs reported to date in filamentous pathogen effector proteins. Some of these motifs appear to define domains that mediate translocation inside host cells.