Serotype Distribution, Antimicrobial Susceptibility, Multilocus Sequencing Type and Virulence of Invasive Streptococcus pneumoniae in China: A Six-Year Multicenter Study

Background:Streptococcus pneumoniae is an important human pathogen that can cause severe invasive pneumococcal diseases (IPDs). The aim of this multicenter study was to investigate the serotype and sequence type (ST) distribution, antimicrobial susceptibility, and virulence of S. pneumoniae strains causing IPD in China.Methods: A total of 300 invasive S. pneumoniae isolates were included in this study. The serotype, ST, and antimicrobial susceptibility of the strains, were determined by the Quellung reaction, multi-locus sequence typing (MLST) and broth microdilution method, respectively. The virulence level of the strains in the most prevalent serotypes was evaluated by a mouse sepsis model, and the expression level of well-known virulence genes was measured by RT-PCR.Results: The most common serotypes in this study were 23F, 19A, 19F, 3, and 14. The serotype coverages of PCV7, PCV10, PCV13, and PPV23 vaccines on the strain collection were 42.3, 45.3, 73.3 and 79.3%, respectively. The most common STs were ST320, ST81, ST271, ST876, and ST3173. All strains were susceptible to ertapenem, levofloxacin, moxifloxacin, linezolid, and vancomycin, but a very high proportion (>95%) was resistant to macrolides and clindamycin. Based on the oral, meningitis and non-meningitis breakpoints, penicillin non-susceptible Streptococcus pneumoniae (PNSP) accounted for 67.7, 67.7 and 4.3% of the isolates, respectively. Serotype 3 strains were characterized by high virulence levels and low antimicrobial-resistance rates, while strains of serotypes 23F, 19F, 19A, and 14, exhibited low virulence and high resistance rates to antibiotics. Capsular polysaccharide and non-capsular virulence factors were collectively responsible for the virulence diversity of S. pneumoniae strains.Conclusion: Our study provides a comprehensive insight into the epidemiology and virulence diversity of S. pneumoniae strains causing IPD in China.

Phylogenetic Diversity and Mycotoxin Potential of Emergent Phytopathogens within the Fusarium tricinctum Species Complex

Recent studies on multiple continents indicate members of the Fusarium tricinctum species complex (FTSC) are emerging as prevalent pathogens of small-grain cereals, pulses, and other economically important crops. These understudied fusaria produce structurally diverse mycotoxins, among which enniatins (ENNs) and moniliformin (MON) are the most frequent and of greatest concern to food and feed safety. Herein a large survey of fusaria in the Fusarium Research Center and Agricultural Research Service culture collections was undertaken to assess species diversity and mycotoxin potential within the FTSC. A 151-strain collection originating from diverse hosts and substrates from different agroclimatic regions throughout the world was selected from 460 FTSC strains to represent the breadth of FTSC phylogenetic diversity. Evolutionary relationships inferred from a 5-locus dataset, using maximum likelihood and parsimony, resolved the 151 strains as 24 phylogenetically distinct species, including nine that are new to science. Of the five genes analyzed, nearly full-length phosphate permease sequences contained the most phylogenetically informative characters, establishing its suitability for species-level phylogenetics within the FTSC. Fifteen of the species produced ENNs, MON, the sphingosine analog 2-amino-14,16- dimethyloctadecan-3-ol (AOD), and the toxic pigment aurofusarin (AUR) on a cracked corn kernel substrate. Interestingly, the five earliest diverging species in the FTSC phylogeny (i.e., F. iranicum, F. flocciferum, F. torulosum, Fusarium spp. FTSC 8 and 24) failed to produce AOD and MON, but synthesized ENNs and/or AUR. Moreover, our reassessment of nine published phylogenetic studies on the FTSC identified 11 additional novel taxa, suggesting this complex comprises at least 36 species.

The pathogenic biomarker alcohol dehydrogenase protein is involved in Bacillus cereus virulence and survival against host innate defence

Bacillus cereus is a spore forming bacteria recognized among the leading agents responsible for foodborne outbreaks in Europe. B. cereus is also gaining notoriety as an opportunistic human pathogen inducing local and systemic infections. The real incidence of such infection is likely underestimated and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We have recently analyzed a large strain collection of varying pathogenic potential. Screening for biomarkers to differentiate among clinical and non-clinical strains, a gene encoding an alcohol dehydrogenase-like protein was identified among the leading candidates. This family of proteins has been demonstrated to be involved in the virulence of several bacterial species. The relevant gene was knocked out to elucidate its function with regards to resistance to host innate immune response, both in vitro and in vivo. Our results demonstrate that the adhB gene plays a significant role in resistance to nitric oxide and oxidative stress in vitro, as well as its pathogenic ability with regards to in vivo toxicity. These properties may explain the pathogenic potential of strains carrying this newly identified virulence factor.

Prevalence and correlates of phenazine resistance in culturable bacteria from a dryland wheat field

Phenazines are a class of bacterially-produced redox-active natural antibiotics that have demonstrated potential as a sustainable alternative to traditional pesticides for the biocontrol of fungal crop diseases. However, the prevalence of bacterial resistance to agriculturally-relevant phenazines is poorly understood, limiting both the understanding of how these molecules might shape rhizosphere bacterial communities and the ability to perform risk assessment for off-target effects. Here, we describe profiles of susceptibility to the antifungal agent phenazine-1-carboxylic acid (PCA) across more than 100 bacterial strains isolated from a wheat field where PCA producers are indigenous and abundant. We find that Gram-positive bacteria are typically more sensitive to PCA than Gram-negative bacteria, but that there is also significant variability in susceptibility both within and across phyla. Phenazine-resistant strains are more likely to be isolated from the wheat rhizosphere, where PCA producers are also more abundant, compared to bulk soil. Furthermore, PCA toxicity is pH-dependent for most susceptible strains and broadly correlates with PCA reduction rates, suggesting that uptake and redox-cycling are important determinants of phenazine toxicity. Our results shed light on which classes of bacteria are most likely to be susceptible to phenazine toxicity in acidic or neutral soils. In addition, the taxonomic and phenotypic diversity of our strain collection represents a valuable resource for future studies on the role of natural antibiotics in shaping wheat rhizosphere communities.

Development of an agar-plug cultivation system for bioactivity assays of actinomycete strain collections

Natural products are an important source of lead compounds for the development of drug substances. Actinomycetes have been valuable especially for the discovery of antibiotics. Increasing occurrence of antibiotic resistance among bacterial pathogens has revived the interest in actinomycete natural product research. Actinobacteria produce a different set of natural products when cultivated on solid growth media compared with submersed culture. Bioactivity assays involving solid media (e.g. agar-plug assays) require manual manipulation of the strains and agar plugs. This is less convenient for the screening of larger strain collections of several hundred or thousand strains. Thus, the aim of this study was to develop a 96-well microplate-based system suitable for the screening of actinomycete strain collections in agar-plug assays. We developed a medium-throughput cultivation and agar-plug assay workflow that allows the convenient inoculation of solid agar plugs with actinomycete spore suspensions from a strain collection, and the transfer of the agar plugs to petri dishes to conduct agar-plug bioactivity assays. The development steps as well as the challenges that were overcome during the development (e.g. system sterility, handling of the agar plugs) are described. We present the results from one exemplary screening campaign targeted to identify compounds inhibiting Agr-based quorum sensing where the workflow was used successfully. We present a novel and convenient workflow to combine agar diffusion assays with microtiter-plate-based cultivation systems in which strains can grow on a solid surface. This workflow facilitates and speeds up the initial medium throughput screening of natural product-producing actinomycete strain collections against monitor strains in agar-plug assays.

A standardized genome architecture for bacterial synthetic biology (SEGA)

Chromosomal recombinant gene expression offers a number of advantages over plasmid-based synthetic biology. However, the methods applied for bacterial genome engineering are still challenging and far from being standardized. Here, in an attempt to realize the simplest recombinant genome technology imaginable and facilitate the transition from recombinant plasmids to genomes, we create a simplistic methodology and a comprehensive strain collection called the Standardized Genome Architecture (SEGA). In its simplest form, SEGA enables genome engineering by combining only two reagents: a DNA fragment that can be ordered from a commercial vendor and a stock solution of bacterial cells followed by incubation on agar plates. Recombinant genomes are identified by visual inspection using green-white colony screening akin to classical blue-white screening for recombinant plasmids. The modular nature of SEGA allows precise multi-level control of transcriptional, translational, and post-translational regulation. The SEGA architecture simultaneously supports increased standardization of genetic designs and a broad application range by utilizing well-characterized parts optimized for robust performance in the context of the bacterial genome. Ultimately, its adaption and expansion by the scientific community should improve predictability and comparability of experimental outcomes across different laboratories.

Identification and Antibiotic Profiling of Wohlfahrtiimonas chitiniclastica, an Underestimated Human Pathogen

In the past 12 years, several case reports have clearly demonstrated that Wohlfahrtiimonas chitiniclastica is capable of causing sepsis and bacteremia in humans. However, since most clinicians are not familiar with this species, little is known about its pathogenicity and treatment options while it is as rare but underestimated human pathogen. Therefore, a larger strain collection is required so that methods can be identified that are most suitable to obtain rapid and reliable identification. Moreover, the antimicrobial resistance profile needs to be elucidated in order to explore possible treatment options. Over a period of 6 years, we therefore have collected a total of 14 W. chitiniclastica isolates in routine diagnostics, which now served as the basis for a comprehensive characterization with respect to identification and antibiotic profiling. We compared the accuracy and convenience of several identification techniques in which MALDI-TOF MS and sequencing of the 16S rRNA gene have proven to be suitable for identification of W. chitiniclastica. In addition, whole genome sequencing (WGS)-based digital DNA-DNA hybridization (dDDH) was used as a reference method for strain identification, and surprised with the detection of a novel W. chitiniclastica subspecies. A combination of in silico and in vitro analyses revealed a first insight into the antimicrobial resistance profile and the molecular basis of antimicrobial resistance. Based on our findings, trimethoprim/sulfamethoxazole, levofloxacin, and cephalosporins (e.g., ceftazidime) may be the best antibiotics to use in order to treat infections caused by W. chitiniclastica, while resistance to fosfomycin, amikacin and tobramycin is observed.

Metal nanoparticle antibacterial effect оn antibiotic-resistant strains of bacteria

The rapid formation of microbial resistance to modern antibacterial drugs requires to search for new, alternative therapies. It is known that some organisms, such as plants, algae, fungi, are able to convert inorganic metal ions into metal nanoparticles due to the recovery process carried out by proteins, sugars and metabolites contained in the tissues and cells of these organisms. At the same time, many plants (e.g., plantain, yarrow, wormwood, turmeric long, calendula, marsh bagulnik, etc.) and metals (copper, silver, gold, zinc, etc.) themselves have antibacterial properties, so that metal nanoparticles obtained by biological method, or via “Green” synthesis method, from extracts of such plants can become a current alternative to many modern antibacterial drugs. The antibacterial mechanism of action of nanoparticles depends on the type of microorganisms affected, as well as on the type of nanoparticles, their concentration, size, and how they are obtained. Based on this, the study of the antibacterial effect of nanoparticles is one of the promising directions of solving the problem of microbial antibiotic resistance. There was examined antibacterial effect of metal nanoparticles containing silver, copper and gold obtained by biological method from the salts of AgNO3, CuSO4, H[AuCl4] metals, respectively, and the extract of the plant — turmeric long (lat. Curcuma longa) — related to the following bacteria strain collection: E. coli (ATCC 25922), S. aureus (ATCC 25923), MRSA (ATCC 38591) and polyresistant clinical strains isolated from patients of the Regional clinical hospital (Krasnoyarsk) — К. рneumoniae, strain 104, P. аeruginosa, strain 40, P. аeruginosa, strain 215, А. baumannii, strain 210, А. baumannii, strain 211. Study allowed to identify the minimum suppressive concentration of nanoparticles by the method of serial dilutions (MUK 4.2.1890-04) with azurin dye. It was proved that metal nanoparticles exhibit different antibacterial efficacy depending on the type of nanometals used and bacterial cultures. Copper nanoparticles have the highest antibacterial activity, and gold nanoparticles have the lowest. The most marked antibacterial effect was observed against clinical polyresistant strains. Metal nanoparticles can become an alternative to the currently known antibacterial drugs, but despite the high efficiency of nanoparticles against polyresistant to antibacterial drugs microorganisms in vitro, it is necessary to take into account their possible toxic effect on live tissues, which requires further study in experiments in vivo.

Lacticaseibacillus paracasei NK112 mitigates Escherichia coli-induced depression and cognitive impairment in mice by regulating IL-6 expression and gut microbiota

The gut microbiota communicates with the brain through microbiota-gut-brain (MGB) and hypothalamus-pituitary-adrenal (HPA) axes and other pathways. Excessive expression of interleukin (IL)-6 is closely associated with the occurrence of the psychiatric disorders depression and dementia. Therefore, to understand whether IL-6 expression-suppressing probiotics could alleviate psychiatric disorders, we isolated IL-6 expression-inhibiting Lacticaseibacillus paracasei (formerly Lactobacillus paracasei) NK112 from the human faecal bacteria strain collection (Neurobiota Research Center, Seoul, Korea) and examined its therapeutic effect for the depression and cognitive impairment in mice. C57 BL/6J mice with depression and cognitive impairment were prepared by exposure to Escherichia coli K1. Oral gavage of NK112 significantly alleviated K1-induced anxious, depressive, and memory-impaired behaviours in the elevated plus maze, tail-suspension and Y-maze tasks, IL-1β, IL-6, and tumour necrosis factor (TNF)-α expression, and nuclear factor kappa beta (NF-κB) activation in the hippocampus, while K1-suppressed brain-derived neurotrophic factor (BDNF) expression increased. Treatment with NK112 also improved K1-induced myeloperoxidase activity, IL-6 and TNF-α expression, and NF-κB activation in the colon and reduced K1-induced Proteobacteria population in the gut microbiota. Heat-killed NK112 and its lysate supernatant, and precipitate fractions also improved anxiety/depression, cognitive impairment, and colitis in mice. In conclusion, NK112, even if heat-killed or lysed, alleviated K1 stress-induced colitis, anxiety/depression, and cognitive impairment by suppressing IL-6, TNF-α, and BDNF expression through the regulation of gut microbiota and NF-κB activation.

Biological properties of swine vesicular disease virus strain 2348 Italy/2008

Swine vesicular disease (SVD) is a viral infectious disease, which, if acute, is manifested by the clinical pattern similar to a number of vesicular diseases including foot-and-mouth disease. In case of subclinical disease, there are no evident clinical signs, therefore the diagnosis is problematic, and there can be the risk of the disease introduction into the Russian Federation with the infected pigs. The key measure for the prevention of SVD introduction involves control diagnostic testing of all animals imported in the country that makes it necessary to keep updated the currently used methods and tools for the disease laboratory diagnosis. The paper demonstrates data on experimental infection of pigs with SVDV strain 2348 Italy/2008 that belongs to the most recent one of the four known phylogenetic groups. The virus was kindly provided by the World Reference Laboratory for Foot-and-Mouth Disease (Pirbright, Great Britain), and it was adapted to the monolayer continuous cell cultures of porcine origin (IB-RS-2 and PGSK-30). The pigs were intradermally infected with concentrated cultured virus at a dose of 109 TCID50. The infected animals demonstrated clinical signs typical for the acute disease. There was evidence that the virus was not transmitted to the intact animal in case husbandry conditions were met that allowed to avoid the infection transmission by the fecal-oral and contact mechanisms. As a result of the experiment, reference sera were collected at different time intervals post infection and their activity was determined using virus microneutralization test in cell culture and ELISA. Aphthae collected from the infected animals were deposited into the Strain collection of the Reference Laboratory for Foot-and-Mouth Disease, FGBI “ARRIAH”.