Respective Parental Strains Research Articles

Resistance risk and mechanism of Ustilaginoidea virens to pydiflumetofen

Rice false smut, caused by Ustilaginoidea virens, is a devastating fungal disease in rice that not only leads to yield reduction but also poses a serious threat to food safety and human health due to the production of numerous mycotoxins. Pydiflumetofen, one of the most promising SDHI fungicides widely used for controlling various plant diseases, lacks available information regarding its antifungal activity against U. virens and the potential risk of resistance development in this pathogen. In this study, we evaluated the sensitivity of 33 field-isolated strains of U. virens to pydiflumetofen using mycelial growth inhibition method and assessed the potential for resistance development. The EC50 values for pydiflumetofen against the tested strains ranged from 0.0032 to 0.0123 μg/mL, with an average EC50 value of 0.0056 ± 0.0025 μg/mL. In addition, four strains of U. virens were randomly selected for chemical taming to evaluate their resistance risk to pydiflumetofen, resulting in the successful generation of eight stable and inheritable resistant mutants at a frequency of 1 %. These mutants exhibited significant differences in biological fitness compared to their respective parental strains. Cross-resistance tests revealed a correlation between pydiflumetofen and fluxapyroxad as well as fluopyram, but no evidence of cross-resistance was observed between pydiflumetofen and boscalid or tebuconazole. Therefore, we can conclude that the risk of resistance development in U. virens to pydiflumetofen is moderate. Finally, the target genes SDHB, SDHC, and SDHD in U. virens were initially identified, cloned, and sequenced to elucidate the mechanism underlying U. virens resistance to pydiflumetofen. Three mutation genotypes were found in the mutants: SDHB-H239Y, SDHB-H239L, and SDHC-A77V. The mutants carrying SDHB-H239Y exhibited low resistance, while SDHC-A77V showed moderate resistance, but the mutants with SDHB-H239L demonstrated high resistance. These findings contribute significantly to our comprehensive understanding of molecular mechanisms involved in the resistance of U. virens to pydiflumetofen, and provide an important reference for chemical control strategies against rice false smut in the field.

Deletion of an immune evasion gene, steD, from a live Salmonella enterica serovar Typhimurium vaccine improves vaccine responses in aged mice.

Non-typhoidal Salmonella (NTS) generally causes self-limiting gastroenteritis. However, older adults (≥65 years) can experience more severe outcomes from NTS infection. We have previously shown that a live attenuated S. Typhimurium vaccine, CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA), was immunogenic in adult but not aged mice. Here we describe modification of CVD 1926 through deletion of steD, a Salmonella effector responsible for host immune escape, which we hypothesized would increase immunogenicity in aged mice. Mel Juso and/or mutuDC cells were infected with S. Typhimurium I77, CVD 1926, and their respective steD mutants, and the MHC-II levels were evaluated. Aged (18-month-old) C57BL/6 mice received two doses of PBS, CVD 1926, or CVD 1926 ΔsteD perorally (109 CFU) and the number of FliC-specific CD4+ T cells were determined. Lastly, aged C57BL/6 mice received three doses of PBS, CVD 1926, or CVD 1926 ΔsteD perorally (109 CFU) and then were challenged perorally with wild-type S. Typhimurium SL1344 (108 CFU). These animals were also evaluated for antibody responses. MHC-II induction was higher in cells treated with steD mutants, compared to their respective parental strains. Compared to PBS-vaccinated mice, CVD 1926 ΔsteD elicited significantly more FliC-specific CD4+ T cells in the Peyer's Patches. There were no significant differences in FliC-specific CD4+ T cells in the Peyer's patches or spleen of CVD 1926- versus PBS-immunized mice. CVD 1926 and CVD 1926 ΔsteD induced similar serum and fecal anti-core and O polysaccharide antibody titers after three doses. After two immunizations, the proportion of seroconverters for CVD 1926 ΔsteD was 83% (10/12) compared to 42% (5/12) for CVD 1926. Compared to PBS-immunized mice, mice immunized with CVD 1926 ΔsteD had significantly lower S. Typhimurium counts in the spleen, cecum, and small intestine upon challenge. In contrast, there were no differences in bacterial loads in the tissues of PBS-vaccinated and CVD 1926-immunized animals. These data suggest that the steD deletion enhanced the immunogenicity of our live attenuated S. Typhimurium vaccine. Deletion of immune evasion genes could be a potential strategy to improve the immunogenicity of live attenuated vaccines in older adults.

Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains

BackgroundThe current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS.ResultsIn our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27–45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84–224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively.ConclusionWhile additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Heterozygous Spink1 Deficiency Promotes Trypsin-dependent Chronic Pancreatitis in Mice

Heterozygous SPINK1 mutations are strong risk factors for chronic pancreatitis in humans, yet heterozygous disruption of mouse Spink1 yielded no pancreatic phenotype. To resolve this contradiction, we usedCRISPR/Cas9-mediated genome editing to generate heterozygous Spink1-deleted mice (Spink1-KOhet) in the C57BL/6N strain and studied the effect of this allele in trypsin-independent and trypsin-dependent pancreatitis models. We investigated severity of acute pancreatitis and progression to chronic pancreatitis in Spink1-KOhet mice after transient (10 injections) and prolonged (2× 8 injections) cerulein hyperstimulation. We crossed Spink1-KOhet mice with T7D23A and T7D22N,K24R mice that carry strongly autoactivating trypsinogen mutants and exhibit spontaneous chronic pancreatitis. Prolonged but not transient cerulein stimulation resulted in increased intrapancreatic trypsin activity and more severe acute pancreatitis in Spink1-KOhet mice relative to the C57BL/6N control strain. After the acute episode, Spink1-KOhet mice developed progressive disease with chronic pancreatitis-like features, whereas C57BL/6N mice recovered rapidly. Trypsinogen mutant mice carrying the Spink1-KOhet allele exhibited strikingly more severe chronic pancreatitis than the respective parent strains. Heterozygous Spink1 deficiency caused more severe acute pancreatitis after prolonged cerulein stimulation and promoted chronic pancreatitis after the cerulein-induced acute episode, and in two strains of trypsinogen mutant mice with spontaneous disease. In contrast, acute pancreatitis induced with limited cerulein hyperstimulation was unaffected by heterozygous Spink1 deletion, in agreement with recent observations that trypsin activity does not mediate pathologic responses in this model. Taken together, the findings strongly support the notion that loss-of-function SPINK1 mutations inhumans increase chronic pancreatitis risk in a trypsin-dependent manner.

Selection and improvement of Saccharomyces cerevisiae by direct and mass mating for integrated first and second generation (1G + 2G) ethanol production

The integrated 1G + 2G ethanol production process seems to be a feasible strategy considering the Brazilian scenario, in which sugarcane molasses and bagasse are available substrates in biorefineries. However, such process imposes harsh conditions to yeasts, which must be able to simultaneously deal with several stresses from molasses and hydrolysate. In this work, improved hybrid strains of Saccharomyces cerevisiae with higher resistance for an integrated 1G + 2G ethanol production were obtained by hybridization technique. Initially, 663 wild S. cerevisiae strains were screened in microculture growth assays. Seven strains were pre-selected out of 40 based on their stress multi-tolerance traits. Subsequently, these pre-selected strains underwent evaluation in cell-recycling fed-batch fermentation conditions utilizing a medium containing molasses-bagasse hydrolysate. The two most successful strains from this evaluation were subjected to sporulation, yielding haploid cells. These haploid cells were then subjected to crossbreeding, employing direct and mass mating techniques. From the initial pool of 7 pre-selected strains, 3 hybrid strains were selected due to their enhanced resistance under the demanding conditions of cell recycling fed-batch fermentation, particularly when utilizing a higher osmotic molasses-hydrolysate medium. These hybrid strains consistently exhibited high cell viability rates and effectively accumulated elevated levels of trehalose and glycogen over the course of five fermentation cycles. Remarkably, they maintained ethanol productivity at levels comparable to their respective parental strains. The genotyping molecular identification attested the difference between the hybrids and the parental strains. The 3 hybrids selected seem to present valuable backgrounds for metabolic engineering for lignocellulosic-derived xylose fermentation.

MicroPET Imaging Assessment of Brain Tau and Amyloid Deposition in 6 × Tg Alzheimer's Disease Model Mice.

Alzheimer’s disease (AD) is characterized by the deposition of extracellular amyloid plaques and intracellular accumulation of neurofibrillary tangles (NFT). Amyloid beta (Aβ) and tau imaging are widely used for diagnosing and monitoring AD in clinical settings. We evaluated the pathology of a recently developed 6 × Tg − AD (6 × Tg) mouse model by crossbreeding 5 × FAD mice with mice expressing mutant (P301L) tau protein using micro-positron emission tomography (PET) image analysis. PET studies were performed in these 6 × Tg mice using [18F]Flutemetamol, which is an amyloid PET radiotracer; [18F]THK5351 and [18F]MK6240, which are tau PET radiotracers; moreover, [18F]DPA714, which is a translocator protein (TSPO) radiotracer, and comparisons were made with age-matched mice of their respective parental strains. We compared group differences in standardized uptake value ratio (SUVR), kinetic parameters, biodistribution, and histopathology. [18F]Flutemetamol images showed prominent cortical uptake and matched well with 6E10 staining images from 2-month-old 6 × Tg mice. [18F]Flutemetamol images showed a significant correlation with [18F]DPA714 in the cortex and hippocampus. [18F]THK5351 images revealed prominent hippocampal uptake and matched well with AT8 immunostaining images in 4-month-old 6 × Tg mice. Moreover, [18F]THK5351 images were confirmed using [18F]MK6240, which revealed significant correlations in the cortex and hippocampus. Uptake of [18F]THK5351 or [18F]MK6240 was highly correlated with [18F]Flutemetamol in 4-month-old 6 × Tg mice. In conclusion, PET imaging revealed significant age-related uptake of Aβ, tau, and TSPO in 6 × Tg mice, which was highly correlated with age-dependent pathology.

Genomic comparison and phenotypic profiling of small colony variants of Burkholderia pseudomallei.

Burkholderia pseudomallei (B. pseudomallei) is an intracellular pathogen that causes melioidosis, a life-threatening infection in humans. The bacterium is able to form small colony variants (SCVs) as part of the adaptive features in response to environmental stress. In this study, we characterize the genomic characteristics, antimicrobial resistance (AMR), and metabolic phenotypes of B. pseudomallei SCV and wild type (WT) strains. Whole-genome sequence analysis was performed to characterize the genomic features of two SCVs (CS and OS) and their respective parental WT strains (CB and OB). Phylogenetic relationship between the four draft genomes in this study and 19 publicly available genomes from various countries was determined. The four draft genomes showed a close phylogenetic relationship with other genomes from Southeast Asia. Broth microdilution and phenotype microarray were conducted to determine the AMR profiles and metabolic features (carbon utilization, osmolytes sensitivity, and pH conditions) of all strains. The SCV strains exhibited identical AMR phenotype with their parental WT strains. A limited number of AMR-conferring genes were identified in the B. pseudomallei genomes. The SCVs and their respective parental WT strains generally shared similar carbon-utilization profiles, except for D,L-carnitine (CS), g-hydroxybutyric acid (OS), and succinamic acid (OS) which were utilized by the SCVs only. No difference was observed in the osmolytes sensitivity of all strains. In comparison, WT strains were more resistant to alkaline condition, while SCVs showed variable growth responses at higher acidity. Overall, the genomes of the colony morphology variants of B. pseudomallei were largely identical, and the phenotypic variations observed among the different morphotypes were strain-specific.

Can only one physiological trait determinate the adverse effect of green fluorescent protein (GFP) incorporation on Vibrio virulence?

Green fluorescent protein (GFP) has been used extensively for in situ animal studies that follow up bacterial infection under epifluorescence microscopy. It is assumed that GFP is acting as a "neutral" protein with no influence on the bacterial physiology. To verify this hypothesis, the virulence of Vibrio splendidus ME9, Vibrio anguillarum NB10, and their respective GFP-tagged strains ME9-GFP and NB10-GFP (transconjugants) was compared in vitro and tested in vivo towards blue mussel (Mytilus edulis) larvae. Results showed that the incorporation of GFP negatively impacted the growth and swimming motility of NB10 in vitro. Correspondingly, the mRNA levels of genes involved in bacterial swimming motility (flaA, flaE, and cheR) were significantly down-regulated in NB10-GFP. As for the strain ME9 on the other hand, GFP incorporation only had a negative effect on swimming motility. However, both the strains NB10-GFP and ME9-GFP showed almost the same virulence as their respective parental strain towards mussel larvae in vivo. Overall, the data presented here demonstrated that incorporation of GFP may cause modifications in cell physiology and highlight the importance of preliminary physiological tests to minimize the negative influence of GFP tagging when it is used to monitor the target localization. The study also supports the idea that the virulence of Vibrio species is determined by complex regulatory networks. Notwithstanding the change of a single physiological trait, especially growth or swimming motility, the GFP-tagged Vibrio strain can thus still be considered usable in studies mainly focusing on the virulence of the strain. KEY POINTS: • The effect of GFP incorporation on physiological trait of Vibrio strains. • The virulence in vibrios could be multifactorial. • The stable virulence of Vibrio strains after GFP incorporation.

A Conserved Role for LRRK2 and Roco Proteins in the Regulation of Mitochondrial Activity.

Parkinson’s Disease (PD) is the second most common neurodegenerative disease world-wide. Mutations in the multidomain protein Leucine Rich Repeat Kinase 2 (LRRK2) are the most frequent cause of hereditary PD. Furthermore, recent data suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in PD pathogenesis. Isolated mitochondria of tissue samples from PD patients carrying LRRK2 mutations display a significant impairment of mitochondrial function. However, due to the complexity of the mitochondrial signaling network, the role of LRRK2 in mitochondrial metabolism is still not well understood. Previously we have shown that D. discoideum Roco4 is a suitable model to study the activation mechanism of LRRK2 in vivo. To get more insight in the LRRK2 pathways regulating mitochondrial activity we used this Roco4 model system in combination with murine RAW macrophages. Here we show that both Dictyostelium roco4 knockout and cells expressing PD-mutants show behavioral and developmental phenotypes that are characteristic for mitochondrial impairment. Mitochondrial activity measured by Seahorse technology revealed that the basal respiration of D. discoideum roco4- cells is significantly increased compared to the WT strain, while the basal and maximal respiration values of cells overexpressing Roco4 are reduced compared to the WT strain. Consistently, LRRK2 KO RAW 264.7 cells exhibit higher maximal mitochondrial respiration activity compared to the LRRK2 parental RAW264.7 cells. Measurement on isolated mitochondria from LRRK2 KO and parental RAW 264.7 cells revealed no difference in activity compared to the parental cells. Furthermore, neither D. discoideum roco4- nor LRRK2 KO RAW 264.7 showed a difference in either the number or the morphology of mitochondria compared to their respective parental strains. This suggests that the observed effects on the mitochondrial respiratory in cells are indirect and that LRRK2/Roco proteins most likely require other cytosolic cofactors to elicit mitochondrial effects.

Manipulation of host ecdysteroid hormone levels facilitates infection by the fungal insect pathogen, Metarhizium rileyi.

Entomopathogenic fungi such as Metarhizium rileyi and Beauveria bassiana are widely used insect biological control agents. Little, however, is known concerning genetic or enzymatic factors that differentiate the mechanisms employed by these two fungal pathogens to infect target hosts. Infection by either of these organisms is known to increase levels of the growth and molting hormone, ecdysone, which also regulates the expression of a number of innate immune pathways. M. rileyi, but not B. bassiana, has apparently evolved an ecdysteroid-22-oxidase (MrE22O) that inactivate ecdysone. We show that deletion of MrE22O impaired virulence compared with the wild-type strain, with an increase in ecdysone titer seen in hosts that was coupled to an increase in the expression of antimicrobial genes. An M. rileyi strain engineered to overexpress MrE22O (MrE22OOE ), as well as trans-expression in B. bassiana (Bb::MrE220OE ) resulted, in strains displaying enhanced virulence and dampening of host immune responses compared with their respective wild-type parental strains. These results indicate that ecdysone plays an important role in mediating responses to fungal infection and that some insect pathogenic fungi have evolved mechanisms for targeting this hormone as a means for facilitating infection.

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization.

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

O antigen restricts lysogenization of non-O157 Escherichia coli strains by Stx-converting bacteriophage phi24B

Acquisition of new prophages that are able to increase the bacterial fitness by the lysogenic conversion is believed to be an important strategy of bacterial adaptation to the changing environment. However, in contrast to the factors determining the range of bacteriophage lytic activity, little is known about the factors that define the lysogenization host range. Bacteriophage phi24B is the paradigmal model of Stx-converting phages, encoding the toxins of the Shiga-toxigenic E. coli (STEC). This virus has been shown to lysogenize a wide range of E. coli strains that is much broader than the range of the strains supporting its lytic growth. Therefore, phages produced by the STEC population colonizing the small or large intestine are potentially able to lysogenize symbiotic E. coli in the hindgut, and these secondary lysogens may contribute to the overall patient toxic load and to lead to the emergence of new pathogenic STEC strains. We demonstrate, however, that O antigen effectively limit the lysogenization of the wild E. coli strains by phi24B phage. The lysogens are formed from the spontaneous rough mutants and therefore have increased sensitivity to other bacteriophages and to the bactericidal activity of the serum if compared to their respective parental strains.

Spatial memory deficiency early in 6xTg Alzheimer\u2019s disease mouse model

Alzheimer’s disease (AD) is mainly characterized by the deposition of extracellular amyloid plaques and intracellular accumulation of neurofibrillary tangles (NFTs). While the recent 5xFAD AD mouse model exhibits many AD-related phenotypes and a relatively early and aggressive amyloid β production, it does not show NFTs. Here, we developed and evaluated a novel AD mouse model (6xTg-AD, 6xTg) by crossbreeding 5xFAD mice with mice expressing mutant (P301L) tau protein (MAPT). Through behavioral and histopathological tests, we analyzed cognitive changes and neuropathology in 6xTg mice compared to their respective parental strains according to age. Spatial memory deficits occurred in 6xTg mice at 2 months of age, earlier than they occurred in 5xFAD mice. Histopathological data revealed aggressive Aβ42 and p-tau accumulation in 6xTg mice. Microglial activation occurred in the cortex and hippocampus of 6xTg mice beginning at 2 months. In 6xTg model mice, the synaptic loss was observed in the cortex from 4 months of age and in the hippocampus from 6 months of age, and neuronal loss appeared in the cortex from 4 months of age and in the hippocampus 6 months of age, earlier than it is observed in the 5xFAD and JNPL3 models. These results showed that each pathological symptom appeared much faster than in their parental animal models. In conclusion, these novel 6xTg-AD mice might be an advanced animal model for studying AD, representing a promising approach to developing effective therapy.

Identification and characterization of mutations responsible for the \u03b2-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus

Staphylococcus aureus strains that are susceptible to the β-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire β-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0–256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of β-lactam resistance in OS-MRSA.

Exposure to a Manuka Honey Wound Gel Is Associated With Changes in Bacterial Virulence and Antimicrobial Susceptibility.

The use of manuka honey for the topical treatment of wounds has increased worldwide owing to its broad spectrum of activity towards bacteria in both planktonic and biofilm growth modes. Despite this, the potential consequences of bacterial exposure to manuka honey, as may occur during the treatment of chronic wounds, are not fully understood. Here, we describe changes in antimicrobial susceptibility and virulence in a panel of bacteria, including wound isolates, following repeated exposure (ten passages) to sub-inhibitory concentrations of a manuka honey based wound gel. Changes in antibiotic sensitivity above 4-fold were predominantly related to increased vancomycin sensitivity in the staphylococci. Interestingly, Staphylococcus epidermidis displayed phenotypic resistance to erythromycin following passaging, with susceptibility profiles returning to baseline in the absence of further honey exposure. Changes in susceptibility to the tested wound gel were moderate (≤ 1-fold) when compared to the respective parent strain. In sessile communities, increased biofilm eradication concentrations over 4-fold occurred in a wound isolate of Pseudomonas aeruginosa (WIBG 2.2) as evidenced by a 7-fold reduction in gentamicin sensitivity following passaging. With regards to pathogenesis, 4/8 bacteria exhibited enhanced virulence following honey wound gel exposure. In the pseudomonads and S. epidermidis, this occurred in conjunction with increased haemolysis and biofilm formation, whilst P. aeruginosa also exhibited increased pyocyanin production. Where virulence attenuation was noted in a passaged wound isolate of S. aureus (WIBG 1.6), this was concomitant to delayed coagulation and reduced haemolytic potential. Overall, passaging in the presence of a manuka honey wound gel led to changes in antimicrobial sensitivity and virulence that varied between test bacteria.

F-specific coliphage detection by the Bluephage method

F-specific coliphages have been proposed as viral indicators of fecal pollution. These intestinal phages infect cells through the F-pili of the host strains used for their detection, Escherichia. coli HS/FAmp in the US-EPA standard method and Salmonella enterica WG49 in the ISO method.The recently designed Bluephage protocol allows the rapid detection of as low as one somatic coliphage in a working day. The current study describes a new Bluephage method designed to exclusively detect F-specific phages. It employs two new host strains, CB14 and CB16, which detect the same number of F-specific phages as their respective parental strains HS and WG49. In the Bluephage method, when the strain is lysed by bacteriophage infection, the yellow medium turns blue. As low as one F-specific phage was detected in 3 to 5 h by this approach and when the sample contained high phage concentrations, results were obtained in less than 3 h. The F-specific Bluephage method can be used with different sample volumes and allows phage quantification by the most probable number technique. Strain CB14 performed more consistently than CB16, with comparable detection efficiency after increasing the incubation time to 50 min without shaking.

Comparison of Fitness Cost and Virulence in Chromosome- and Plasmid-Mediated Colistin-Resistant Escherichia coli.

Five types of Escherichia coli strains were obtained and sequenced: colistin-susceptible (CL-S) strains, in vitro induced colistin-resistant (CL-IR) strains, mcr-1-negative colistin-resistant strains from livestock (CL-chrR), mcr-1-positive colistin-resistant strains (CL-mcrR), and mcr-1-transferred transconjugants (TC-mcr). Amino acid alterations of PmrAB, PhoPQ, and EptA were identified, and their mRNA expression was measured. Their growth rate was evaluated, and an in vitro competition assay was performed. Virulence was compared through serum resistance and survival in macrophages and Drosophila melanogaster. CL-IR and CL-chrR strains were colistin-resistant due to amino acid alterations in PmrAB, PhoPQ, or EptA, and their overexpression. All colistin-resistant strains did not show reduced growth rates compared with CL-S strains. CL-IR and CL-chrR strains were less competitive than the susceptible strain, but CL-mcrR strains were not. In addition, TC-mcr strains were also significantly more competitive than their respective parental susceptible strain. CL-IR strains had similar or decreased survival rates in human serum, macrophages, and fruit flies, compared with their parental, susceptible strains. CL-chrR strains were also less virulent than CL-S strains. Although CL-mcrR strains showed similar survival rates in human serum and fruit fly to CL-S strains, the survival rates of TC-mcr strains decreased significantly in human serum, macrophages, and fruit flies, compared with their susceptible recipient strain (J53). Chromosome-mediated, colistin-resistant E. coli strains have a fitness cost, but plasmids bearing mcr-1 do not increase the fitness burden of E. coli. Along with high usage of polymyxins, the no fitness cost of mcr-1-positive strains may facilitate rapid spread of colistin resistance.

Fusaristatin A production negatively affects the growth and aggressiveness of the wheat pathogen Fusarium pseudograminearum

Fusarium pseudograminearum (Fp), the causative fungal pathogen of the diseases Fusarium crown rot, is an important constraint to cereals production in many countries including Australia. Fp produces a number of secondary metabolites throughout its life cycle. One of these metabolites, the cyclic lipopeptide fusaristatin A, is encoded by a specific gene cluster containing a polyketide synthase and a three-module non-ribosomal peptide synthetase. However, a recent survey of Fp populations across Australia suggests that this cluster may only be present in a subset of isolates from Western Australia (WA). In this study, we screened 319 Fp isolates from WA and 110 Fp isolates from the Australian eastern states of New South Wales, Victoria, Queensland and South Australia to examine the distribution of this gene cluster among Australian Fp populations. The fusaristatin A gene cluster was found to be present in ~50% of Fp isolates from WA but completely absent in Fp isolates from eastern states. To determine its potential function, mutants of the fusaristatin A gene cluster were generated by disrupting the non-ribosomal peptide synthetase and polyketide synthase genes simultaneously in two different parental backgrounds. The mutants showed increased growth rates and were significantly more aggressive than their respective parental strains on wheat in crown rot pathogenicity assays. This suggested that fusaristatin A has a negative effect on fungal development and aggressiveness. The possible reasons for the geographically restricted presence of the fusaristatin A gene cluster and its role in fungal biology are discussed.

Effect of trichodiene synthase encoding gene expression in Trichoderma strains on their effectiveness in the control of Acanthoscelides obtectus

Acanthoscelides obtectus, known as the bean weevil, is an insect pest that feeds on wild and cultivated common beans. Four Trichoderma isolates, two trichodiene (TD) producers (T34–5.27, E20–5.7) and their corresponding parental strains (T34, E20), were evaluated to determine the effect of their metabolites against A. obtectus adults. TD is a non-phytotoxic Volatile Organic Compound (VOC), which is the first commitment intermediate in the biosynthesis of trichothecenes. Accumulated emergence of insects was reduced in beans treated with these fungal isolates, being more reduced with E20 and T34 strains. TD producer strains, which previously showed to produce a higher number of damaged beans than their respective parental strains, gave rise to a higher insects emergence. Beans treated with fungal isolates had a lower weight loss than the control treatment, with T34 exhibiting the lowest weight loss among all fungal isolates analyzed. Furthermore, E20 showed the higher repellent activity against A. obtectus adults and produced a smaller number of holes per bean. Thus, Trichoderma isolates evaluated are good for the biological control of A. obtectus pest on beans, in which the parental strains (E20 and T34) were those exhibiting a better control of this insect than their respective TD-producer derivates strains. These results suggest that production of TD reduces effectiveness of Trichoderma T34 and E20 strains to control this insect pest.

MgrA Governs Adherence, Host Cell Interaction, and Virulence in a Murine Model of Bacteremia Due to Staphylococcus aureus.

MgrA is an important global virulence gene regulator in Staphylococcus aureus. In the present study, the role of mgrA in host-pathogen interactions related to virulence was explored in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. In vitro susceptibilities to human defense peptides (HDPs), adherence to fibronectin (Fn) and endothelial cells (ECs), EC damage, α-toxin production, expression of global regulator (eg, agr RNAIII) and its downstream effectors (eg, α-toxin [hla] and Fn binding protein A [fnbA]), MgrA binding to fnbA promoter, and the effect on HDP-induced mprF and dltA expression were analyzed. The impact of mgrA on virulence was evaluated using a mouse bacteremia model. mgrA mutants displayed significantly higher susceptibility to HDPs, which might be related to the decreased HDP-induced mprF and dltA expression but decreased Fn and EC adherence, EC damage, α-toxin production, agr RNAIII, hla and fnbA expression, and attenuated virulence in the bacteremia model as compared to their respective parental and mgrA-complemented strains. Importantly, direct binding of MgrA to the fnbA promoter was observed. These results suggest that mgrA mediates host-pathogen interactions and virulence and may provide a novel therapeutic target for invasive S. aureus infections.