Bacillus based biocontrol on Brassica

Many bacterial strains have been shown to mediate protection to biotic stress and promote growth of plants. Different bacteria can mediate protection in different ways e.g. by inhibition, competition or increasing plant resistance. Examples of bacteria that mediate protection to plants include different Pseudomonas, Serratia and Bacillus strains. Bacillus strains have one major advantage toward other biocontrol strains and that is the ability to form spores that are resilient against chemicals and mechanical damage. I have studied the effect of four closely related Bacillus strains on plants in two different projects, one concerned with oilseed rape (Brassica napus) and the other using Arabidopsis thaliana to allow mechanistic studies of the interaction. The bacterial strains are all classified as Bacillus amyloliquefaciens. These bacterial strains have been tested for phenological effects on plants and for plant protection towards pathogens like Alternaria brassicae, Botrytis cinerea, Leptosphaeria maculans, and Verticillium longisporum. Production of antifungal compounds by the strains and the effects on the different pathogens were investigated. Two potential candidates for biocontrol were identified. Both Bacillus strains were found to provide significant protection of oilseed rape against the four pathogens. The effects of Bacillus treatment on the B. napus transcriptome were studied using the cDNA-AFLP technique. Bacillus priming had strong systemic effects on leaf transcripts but small effects on roots. This far 65 differentially expressed plant genes have been identified due to Bacillus treatment, of which many seem related to metabolism. The effect of Bacillus seed treatment has also been studied on Arabidopsis. Significant protection was achieved also here using the same two strains toward Alternaria and Leptosphaeria as well as Pseudomonas syringae as pathogens. Arabidopsis signalling mutant studies showed that functional jasmonic acid (JA) and ethylene (Et) signalling as well as Npr1 were needed for Bacillus biocontrol. Expression levels of marker genes depending on these signalling pathways showed no increase upon Bacillus treatment, while an increase of the JA dependent marker occurred after Bacillus treated plants were infected by P. syringae. Altogether, Bacillus primed biocontrol seems to be based on induced systemic resistance (ISR)

In vitro and in vivo selection of potentially probiotic lactobacilli from Nocellara del Belice table olives

Table olives are increasingly recognized as a vehicle as well as a source of probiotic bacteria, especially those fermented with traditional procedures based on the activity of indigenous microbial consortia, originating from local environments. In the present study, we report characterization at the species level of 49 Lactic Acid Bacteria (LAB) strains deriving from Nocellara del Belice table olives fermented with the Spanish or Castelvetrano methods, recently isolated in our previous work. Ribosomal 16S DNA analysis allowed identification of 4 Enterococcus gallinarum, 3 E. casseliflavus, 14 Leuconostoc mesenteroides, 19 Lactobacillus pentosus, 7 L. coryniformis, and 2 L. oligofermentans. The L. pentosus and L. coryniformis strains were subjected to further screening to evaluate their probiotic potential, using a combination of in vitro and in vivo approaches. The majority of them showed high survival rates under in vitro simulated gastro-intestinal conditions, and positive antimicrobial activity against Salmonella enterica serovar Typhimurium, Listeria monocytogenes and enterotoxigenic Escherichia coli (ETEC) pathogens. Evaluation of antibiotic resistance to ampicillin, tetracycline, chloramphenicol, or erythromycin was also performed for all selected strains. Three L. coryniformis strains were selected as very good performers in the initial in vitro testing screens, they were antibiotic susceptible, as well as capable of inhibiting pathogen growth in vitro. Parallel screening employing the simplified model organism Caenorhabditis elegans, fed the Lactobacillus strains as a food source, revealed that one L. pentosus and one L. coryniformis strains significantly induced prolongevity effects and protection from pathogen-mediated infection. Moreover, both strains displayed adhesion to human intestinal epithelial Caco-2 cells and were able to outcompete foodborne pathogens for cell adhesion. Overall, these results are suggestive of beneficial features for novel LAB strains, which renders them promising candidates as starters for the manufacturing of fermented table olives with probiotic added value

Characterising two-pathogen competition in spatially structured environments

Different pathogens spreading in the same host population often generate complex co-circulation dynamics because of the many possible interactions between the pathogens and the host immune system, the host life cycle, and the space structure of the population. Here we focus on the competition between two acute infections and we address the role of host mobility and cross-immunity in shaping possible dominance/co-dominance regimes. Host mobility is modelled as a network of traveling flows connecting nodes of a metapopulation, and the two-pathogen dynamics is simulated with a stochastic mechanistic approach. Results depict a complex scenario where, according to the relation among the epidemiological parameters of the two pathogens, mobility can either be non-influential for the competition dynamics or play a critical role in selecting the dominant pathogen. The characterisation of the parameter space can be explained in terms of the trade-off between pathogen's spreading velocity and its ability to diffuse in a sparse environment. Variations in the cross-immunity level induce a transition between presence and absence of competition. The present study disentangles the role of the relevant biological and ecological factors in the competition dynamics, and provides relevant insights into the spatial ecology of infectious diseases.Comment: 30 pages, 6 figures, 1 table. Final version accepted for publication in Scientific Report

Genetic characterization of clinical and environmental Vibrio parahaemolyticus from the Northeast USA reveals emerging resident and non-indigenous pathogen lineages

Gastric infections caused by the environmentally transmitted pathogen, Vibrio parahaemolyticus, have increased over the last two decades, including in many parts of the United States (US). However, until recently, infections linked to shellfish from the cool northeastern US waters were rare. Cases have risen in the Northeast, consistent with changes in local V. parahaemolyticus populations toward greater abundance or a shift in constituent pathogens. We examined 94 clinical isolates from a period of increasing disease in the region and compared them to 200 environmental counterparts to identify resident and non-indigenous lineages and to gain insight into the emergence of pathogenic types. Genotyping and multi-locus sequence analysis (MLSA) of clinical isolates collected from 2010 to 2013 in Massachusetts, New Hampshire, and Maine revealed their polyphyletic nature. Although 80% of the clinical isolates harbored the trh hemolysin either alone or with tdh, and were urease positive, 14% harbored neither hemolysin exposing a limitation for these traits in pathogen detection. Resident sequence type (ST) 631 strains caused seven infections, and show a relatively recent history of recombination with other clinical and environmental lineages present in the region. ST34 and ST674 strains were each linked to a single infection and these strain types were also identified from the environment as isolates harboring hemolysin genes. Forty-two ST36 isolates were identified from the clinical collection, consistent with reports that this strain type caused a rise in regional infections starting in 2012. Whole-genome phylogenies that included three ST36 outbreak isolates traced to at least two local sources demonstrated that the US Atlantic coastal population of this strain type was indeed derived from the Pacific population. This study lays the foundation for understanding dynamics within natural populations associated with emergence and invasion of pathogenic strain types in the region

Preliminary Analysis of the Anti-biofilm Efficacy of Manuka Honey on Extended Spectrum Β-lactamase Producing Escherichia Coli Tem-3 and Klebsiella Pneumoniae Shv18, Associated with Urinary Tract Infections

open access articleUrinary Tract Infections (UTIs) are one of the most common infections in the UK and many other parts of the world. The prevalence of the Extended Spectrum β-Lactamases (ESBLs) producing UTIs, combined with their ability to form a bio film, has significantly risen and is limiting therapeutic options. This study investigated the anti-bio film activity of Manuka honey on two ESBL producing pathogens, Escherichia coli TEM-3 and Klebsiella pneumonia SHV18, commonly found in UTIs. The ESBL production was confirmed by the double disk synergy method used to confirm the ESBL production. The antibacterial activity of Manuka honey was determined using the agar well diffusion method. The Minimum Inhibitory Concentration (MIC) was established using serially diluted honey ranging from 50% to 1.56%. The effect of Manuka honey on the pathogen bio films was analysed using the Tissue Culture Plate method, with an established MIC and under 24h incubation with the honey. The results indicated that K. pneumonia SHV18 is a stronger bio film producer than E. coli TEM 3. 50% (w/v) MIC Manuka honey appears to fully prevent the plank tonic growth of both strains. A significant reduction of 81% of the E. coli TEM3 (p < 0.001) and 52% of the K. pneumonia SHV18 (p = 0.001) bio film biomass was observed. The E. coli bio films were found to be more sensitive to the 50% (w/v) honey dilution than those produced by K. pneumonia. The study indicated the anti-bio film potency of Manuka honey and its potential to become an alternative treatment for the ESBL producing pathogens associated with UTIs

IL-17 can be protective or deleterious in murine pneumococcal pneumonia

Streptococcus pneumoniae is the major bacterial cause of community-acquired pneumonia, and the leading agent of childhood pneumonia deaths worldwide. Nasal colonization is an essential step prior to infection. The cytokine IL-17 protects against such colonization and vaccines that enhance IL-17 responses to pneumococcal colonization are being developed. The role of IL-17 in host defence against pneumonia is not known. To address this issue, we have utilized a murine model of pneumococcal pneumonia in which the gene for the IL-17 cytokine family receptor, Il17ra, has been inactivated. Using this model, we show that IL-17 produced predominantly from γδ T cells protects mice against death from the invasive TIGR4 strain (serotype 4) which expresses a relatively thin capsule. However, in pneumonia produced by two heavily encapsulated strains with low invasive potential (serotypes 3 and 6B), IL-17 significantly enhanced mortality. Neutrophil uptake and killing of the serotype 3 strain was significantly impaired compared to the serotype 4 strain and depletion of neutrophils with antibody enhanced survival of mice infected with the highly encapsulated SRL1 strain. These data strongly suggest that IL-17 mediated neutrophil recruitment to the lungs clears infection from the invasive TIGR4 strain but that lung neutrophils exacerbate disease caused by the highly encapsulated pneumococcal strains. Thus, whilst augmenting IL-17 immune responses against pneumococci may decrease nasal colonization, this may worsen outcome during pneumonia caused by some strains

Use of Carnobacterium piscicola to limit the growth of Listeria monocytogenes in mussel products : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Bacteria were screened in order to find an organism antagonistic to Listeria monocytogenes which could be applied to mussel products and enhance their safety, especially when temperature-abused. A Listeria monocytogenes isolate from the seafood industry was selected as the target organism. Strains of Lactobacillus reuteri and Enterococcus fecium were screened on plates incubated at 35°C and 10°C for anti-listerial compounds, but none were found. A non-bacteriocinogenic strain of Carnobacterium piscicola, A9b- was selected as the antagonist for detailed examination of growth in broth, agar and mussel systems at 10°C. This temperature was chosen to represent temperature abuse of refrigerated products. To distinguish between the growth of the Carnobacterium piscicola strain and wild-type Listeria monocytogenes a "semi-selective" agar was developed using phenol-red indicator, and mannitol as the sole carbohydrate source. Growth rates of Carnobacterium piscicola and Listeria monocytogenes were compared when grown alone and as a co-culture in agar and broth. Growth rates of Listeria monocytogenes when grown alone, and in the presence of Carnobacterium piscicola, were determined on mussels. Regression analyses were done for the inhibition of Listeria monocytogenes by Carnobacterium piscicola. In all cases Carnobacterium piscicola significantly inhibited the growth of Listeria monocytogenes (P broth = 0.018, P agar <0.001, P mussels < 0.001). Growth of both organisms was faster in broth, than on mussels or agar. The greatest inhibition of Listeria monocytogenes was observed in broth reaching log₁₀4.8 at 41 hours of incubation, prior to decreasing after this time. In agar and mussels the inhibition lasted longer and had not decreased at the end of the trial. The log₁₀ reduction in growth of Listeria monocytogenes in agar was measured at 3.4 and in mussels measured at 1.6. These results were statistically significant (P<0.001 for all). Inhibition of wild type Listeria monocytogenes was also shown in broth when a much lower concentration of Carnobacterium piscicola was used. These results should be considered as preliminary and further confirmatory work should be done. However, Carnobacterium piscicola A9b- shows promise as an antagonistic organism to assist in the control of Listeria monocytogenes in mussel products along with industry-accepted good hygienic practices

The influence of the accessory genome on bacterial pathogen evolution

Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorise the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution