Transcriptomic analysis of Vibrio alginolyticus challenged by Rhizoma coptidis reveals mechanisms of virulence genes.

Rhizoma coptidis, a Chinese herbal medicine widely used to treat various bacterial infections, has the potential to develop antibiotic substitutes to overcome the drug resistance of Vibrio alginolyticus. To study the inhibitory effect of R. coptidis on V. alginolyticus, we sequenced the transcriptomes of three groups of samples of wild-type V. alginolyticus (CK) and V. alginolyticus, which were stressed by 5 mg/mL R. coptidis for 2 h (RC_2 h) and 4 h (RC_4 h). CK was compared with RC_2 h and RC_4 h, respectively, and a total of 1565 differentially expressed genes (DEGs) (988 up-regulated and 577 down-regulated) and 1737 DEGs (1152 up-regulated and 585 down-regulated) were identified. Comparing RC_2 h with RC_4 h, 156 DEGs (114 up-regulated and 42 down-regulated) were identified. The ability of biofilm formation and motility of V. alginolyticus altered upon with different concentrations of R. coptidis. Interestingly, relative expression patterns of virulence genes appeared statistically significantly varied, upon different concentrations of R. coptidis extract. DEGs were annotated to the Gene Ontology (GO) database for function enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the results showed that the main enriched pathways, was those related to the virulence of V. alginolyticus. This study provides a new perspective for understanding the complex pathogenic mechanism of V. alginolyticus. R. coptidis could potnetially be used as alternative or complimnetary to antibiotics to treat infections after further research.

Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani.

Plant pathogens secrete fungal-specific common in several fungal extracellular membrane (CFEM) effectors to manipulate host immunity and contribute to their virulence. Little is known about effectors and their functions in Alternaria solani, the necrotrophic fungal pathogen causing potato early blight. To identify candidate CFEM effector genes, we mined A. solani genome databases. This led to the identification of 12 genes encoding CFEM proteins (termed AsCFEM1-AsCFEM12) and 6 of them were confirmed to be putative secreted effectors. In planta expression revealed that AsCFEM6 and AsCFEM12 have elicitor function that triggers plant defense response including cell death in different botanical families. Targeted gene disruption of AsCFEM6 and AsCFEM12 resulted in a change in spore development, significant reduction of virulence on potato and eggplant susceptible cultivars, increased resistance to fungicide stress, variation in iron acquisition and utilization, and the involvement in 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway. Using maximum likelihood method, we found that positive selection likely caused the polymorphism within AsCFEM6 and AsCFEM12 homologs in different Alternaria spp. Site-directed mutagenesis analysis indicated that positive selection sites within their CFEM domains are required for cell death induction in Nicotiana benthamiana and are critical for response to abiotic stress in yeast. These results demonstrate that AsCFEM effectors possess additional functions beyond their roles in host plant immune response and pathogen virulence.

Molecular Epidemiology and Virulence Gene Analysis of Methicillin-Resistant Staphylococcus aureus Associated with Skin and Soft Tissue Infections in the Shaoxing Region.

Objective: This study aimed to investigate the prevalence, molecular types, and virulence genes of methicillin-resistant Staphylococcus aureus (MRSA) causing skin and soft tissue infections (SSTIs) in the Shaoxing region. Methods: MRSA strains were collected from patients with SSTIs in Shaoxing People's Hospital from January 2019 to December 2019. We conducted SCCmec typing, Staphylococcus protein A (SPA) typing, multilocus sequence typing (MLST), and virulence gene analysis using whole-genome sequencing on all MRSA strains. Results: The detection rate of community-acquired MRSA (CA-MRSA) isolated from SSTI patients in our hospital was 33.3% (6/18). The primary SCCmec types of CA-MRSA strains were IV and V, with IVg(2B) and V(5C2&5) accounting for 16.7% each. Hospital-acquired MRSA (HA-MRSA) strains primarily exhibited SCCmec types IVa(2B) (25.0%), followed by II(2A) (16.7%), V(5C2) (16.7%), and V(5C2&5) (8.3%). SPA typing indicated that CA-MRSA strains causing SSTIs were predominantly t437 (14.3%), t034 (14.3%), t309 (14.3%), t4549 (14.3%), and t7637 (14.3%). The primary SPA type of HA-MRSA strains was t311 (16.7%). MLST typing revealed that the main sequence types (STs) of CA-MRSA strains causing SSTIs were ST22 (33.3%), followed by ST398, ST59, ST88, and ST630, each accounting for 16.7%. The principal STs of HA-MRSA strains were ST398 (16.7%), ST59 (16.7%), ST88 (16.7%), and ST5 (16.7%), followed by ST22, ST630, ST6, and ST188, each at 8.3%. The primary clones of CA-MRSA strains causing SSTIs were ST59-t437-IVg(2B) (16.7%) and ST630-t4549-V(5C2&5) (16.7%), while the primary clones of HA-MRSA strains were ST59-t437-IVa(2B), ST630-t4549-V(5C2&5), ST6-t304-IVa(2B), ST5-t311-II(2A), ST59-t172-IVa(2B), ST398-t571-V(5C2), ST398-t034-V(5C2), and ST5-t311-II(2A), each accounting for 8.3%. The detection rate of the lukSF-PV virulence gene was higher in CA-MRSA strains (50.0%) than in HA-MRSA strains (16.7%). Conclusions: The isolation rate of CA-MRSA strains causing SSTIs was high in Shaoxing People's Hospital, with ST59-t437-IVg(2B) and ST630-t4549-V(5C2&5) being the predominant clones. MRSA strains exhibited multiple virulence genes, with the lukSF-PV gene having a higher detection rate in CA-MRSA strains, signifying its importance as a virulence factor in CA-MRSA.

The Effect of Sub-MIC of Salvadora persica on Growth and Virulence Gene Expressions in Streptococcus mutans.

Dental caries is a multifactorial infectious chronic disease caused by particular bacteria and their virulence products that causes demineralization and progressive deterioration of the dental enamel. Many studies have proven miswak to have a critical antibacterial impact, particularly on cariogenic bacteria and periodontal pathogens, in the oral cavity. This study aimed to investigate the effect of different concentrations of Salvadora persica plant extract on growth and virulence gene expressions at mRNA levels in S. mutans. A total of 191 clinical samples from tooth swabs were collected, and sub-cultured on specific medium agar identified using biochemical and molecular approaches. MIC for the extract was determined and a bacterial growth curve was made to determine the growth phases and the optimum time for adding the extract at different concentrations. RT-qPCR technique was performed, and the REST-2009 software program was used for data analysis. Out of 191 swabs from the tooth 31 isolates were identified using several biochemical and molecular tests. Several S. mutans biofilm-related virulence genes and their Ct values were produced from RT-PCR under the effect of low and high doses of Meswak concentrations. Ct values and reaction efficiency were produced in RT-qPCR by Rotorgen3000, data then were analysed by REST-2009 software. Five isolates were selected to examine the effect of the extract on the mRNA levels using qPCR after growing them with both doses of the extract for about 30hrs. Levels of virulence gene mRNA were regulated differentially in cultures with added both extract doses. The isolates produced significantly lower virulence gene mRNA levels in cultures grown with both plant extract doses. The results produced in this study here provide new insights regarding several virulence gene expressions in S. mutans at the molecular levels when grown under different concentrations of Salvadora persica plant extract.

Diversity of virulence and antibiotic resistance genes expressed in Class A biosolids and biosolids-amended soil as revealed by metatranscriptomic analysis.

Class A biosolids is a treated sewage sludge, commonly applied to agricultural fields, home lawns/gardens, golf courses, forests, and remediation sites around the world. This practice is of public and agricultural concern due to the possibility that biosolids contain antibiotic-resistant bacteria and fungal pathogens that could persist for extended periods in soil. This possibility was determined by metatranscriptomic analysis of virulence, antibiotic resistance, and plasmid conjugation genes, a Class A biosolids, organically managed soil, and biosolids-amended soil under realistic conditions. Biosolids harbored numerous transcriptionally active pathogens, antibiotic resistance genes, and conjugative genes that annotated mostly to Gram-positive pathogens of animal hosts. Biosolids amendment to soil significantly increased the expression of virulence genes by numerous pathogens and antibiotic-resistant genes that were strongly associated with biosolids. Biosolids amendment also significantly increased the expression of virulence genes by native soil fungal pathogens of plant hosts, which suggests higher risks of crop damage by soil fungal pathogens in biosolids-amended soil. Although results are likely to be different in other soils, biosolids, and microbial growth conditions, they provide a more holistic, accurate view of potential health risks associated with biosolids and biosolids-amended soils than has been achievable with more selective cultivation and PCR-based techniques.

The adcA and lmb Genes Play an Important Role in Drug Resistance and Full Virulence of Streptococcus suis.

Streptococcus suis is an recognized zoonotic pathogen of swine and severely threatens human health. Zinc is the second most abundant transition metal in biological systems. Here, we investigated the contribution of zinc to the drug resistance and pathogenesis of S. suis. We knocked out the genes of AdcACB and Lmb, two Zn-binding lipoproteins. Compared to the wild-type strain, we found that the survival rate of this double-mutant strain (ΔadcAΔlmb) was reduced in Zinc-limited medium, but not in Zinc-supplemented medium. Additionally, phenotypic experiments showed that the ΔadcAΔlmb strain displayed impaired adhesion to and invasion of cells, biofilm formation, and tolerance of cell envelope-targeting antibiotics. In a murine infection model, deletion of the adcA and lmb genes in S. suis resulted in a significant decrease in strain virulence, including survival rate, tissue bacterial load, inflammatory cytokine levels, and histopathological damage. These findings show that AdcA and Lmb are important for biofilm formation, drug resistance, and virulence in S. suis. IMPORTANCE Transition metals are important micronutrients for bacterial growth. Zn is necessary for the catalytic activity and structural integrity of various metalloproteins involved in bacterial pathogenic processes. However, how these invaders adapt to host-imposed metal starvation and overcome nutritional immunity remains unknown. Thus, pathogenic bacteria must acquire Zn during infection in order to successfully survive and multiply. The host uses nutritional immunity to limit the uptake of Zn by the invading bacteria. The bacterium uses a set of high-affinity Zn uptake systems to overcome this host metal restriction. Here, we identified two Zn uptake transporters in S. suis, AdcA and Lmb, by bioinformatics analysis and found that an adcA and lmb double-mutant strain could not grow in Zn-deficient medium and was more sensitive to cell envelope-targeting antibiotics. It is worth noting that the Zn uptake system is essential for biofilm formation, drug resistance, and virulence in S. suis. The Zn uptake system is expected to be a target for the development of novel antimicrobial therapies.

Multiomics analysis reveals the molecular mechanisms underlying virulence in Rhizoctonia and jasmonic acid-mediated resistance in Tartary buckwheat (Fagopyrum tataricum).

Rhizoctonia solani is a devastating soil-borne pathogen that seriously threatens the cultivation of economically important crops. Multiple strains with a very broad host range have been identified, but only 1 (AG1-IA, which causes rice sheath blight disease) has been examined in detail. Here, we analyzed AG4-HGI 3 originally isolated from Tartary buckwheat (Fagopyrum tataricum), but with a host range comparable to AG1-IA. Genome comparison reveals abundant pathogenicity genes in this strain. We used multiomic approaches to improve the efficiency of screening for disease resistance genes. Transcriptomes of the plant-fungi interaction identified differentially expressed genes associated with virulence in Rhizoctonia and resistance in Tartary buckwheat. Integration with jasmonate-mediated transcriptome and metabolome changes revealed a negative regulator of jasmonate signaling, cytochrome P450 (FtCYP94C1), as increasing disease resistance probably via accumulation of resistance-related flavonoids. The integration of resistance data for 320 Tartary buckwheat accessions identified a gene homolog to aspartic proteinase (FtASP), with peak expression following R. solani inoculation. FtASP exhibits no proteinase activity but functions as an antibacterial peptide that slows fungal growth. This work reveals a potential mechanism behind pathogen virulence and host resistance, which should accelerate the molecular breeding of resistant varieties in economically essential crops.

Genomes of four Streptomyces strains reveal insights into putative new species and pathogenicity of scab-causing organisms.

Genomes of four Streptomyces isolates, two putative new species (Streptomyces sp. JH14 and Streptomyces sp. JH34) and two non thaxtomin-producing pathogens (Streptomyces sp. JH002 and Streptomyces sp. JH010) isolated from potato fields in Colombia were selected to investigate their taxonomic classification, their pathogenicity, and the production of unique secondary metabolites of Streptomycetes inhabiting potato crops in this region. The average nucleotide identity (ANI) value calculated between Streptomyces sp. JH34 and its closest relatives (92.23%) classified this isolate as a new species. However, Streptomyces sp. JH14 could not be classified as a new species due to the lack of genomic data of closely related strains. Phylogenetic analysis based on 231 single-copy core genes, confirmed that the two pathogenic isolates (Streptomyces sp. JH010 and JH002) belong to Streptomyces pratensis and Streptomyces xiamenensis, respectively, are distant from the most well-known pathogenic species, and belong to two different lineages. We did not find orthogroups of protein-coding genes characteristic of scab-causing Streptomycetes shared by all known pathogenic species. Most genes involved in biosynthesis of known virulence factors are not present in the scab-causing isolates (Streptomyces sp. JH002 and Streptomyces sp. JH010). However, Tat-system substrates likely involved in pathogenicity in Streptomyces sp. JH002 and Streptomyces sp. JH010 were identified. Lastly, the presence of a putative mono-ADP-ribosyl transferase, homologous to the virulence factor scabin, was confirmed in Streptomyces sp. JH002. The described pathogenic isolates likely produce virulence factors uncommon in Streptomyces species, including a histidine phosphatase and a metalloprotease potentially produced by Streptomyces sp. JH002, and a pectinesterase, potentially produced by Streptomyces sp. JH010. Biosynthetic gene clusters (BGCs) showed the presence of clusters associated with the synthesis of medicinal compounds and BGCs potentially linked to pathogenicity in Streptomyces sp. JH010 and JH002. Interestingly, BGCs that have not been previously reported were also found. Our findings suggest that the four isolates produce novel secondary metabolites and metabolites with medicinal properties.

Hypothetical Protein VDAG_07742 Is Required for Verticillium dahliae Pathogenicity in Potato.

Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillium wilt in host plants, a particularly serious problem in potato cultivation. Several pathogenicity-related proteins play important roles in the host infection process, hence, identifying such proteins, especially those with unknown functions, will surely aid in understanding the mechanism responsible for the pathogenesis of the fungus. Here, tandem mass tag (TMT) was used to quantitatively analyze the differentially expressed proteins in V. dahliae during the infection of the susceptible potato cultivar "Favorita". Potato seedlings were infected with V. dahliae and incubated for 36 h, after which 181 proteins were found to be significantly upregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that most of these proteins were involved in early growth and cell wall degradation. The hypothetical, secretory protein with an unknown function, VDAG_07742, was significantly upregulated during infection. The functional analysis with knockout and complementation mutants revealed that the associated gene was not involved in mycelial growth, conidial production, or germination; however, the penetration ability and pathogenicity of VDAG_07742 deletion mutants were significantly reduced. Therefore, our results strongly indicate that VDAG_07742 is essential in the early stage of potato infection by V. dahliae.

Genome-scale model of Pseudomonas aeruginosa metabolism unveils virulence and drug potentiation.

Pseudomonas aeruginosa is one of the leading causes of hospital-acquired infections. To decipher the metabolic mechanisms associated with virulence and antibiotic resistance, we have developed an updated genome-scale model (GEM) of P. aeruginosa. The model (iSD1509) is an extensively curated, three-compartment, and mass-and-charge balanced BiGG model containing 1509 genes, the largest gene content for any P. aeruginosa GEM to date. It is the most accurate with prediction accuracies as high as 92.4% (gene essentiality) and 93.5% (substrate utilization). In iSD1509, we newly added a recently discovered pathway for ubiquinone-9 biosynthesis which is required for anaerobic growth. We used a modified iSD1509 to demonstrate the role of virulence factor (phenazines) in the pathogen survival within biofilm/oxygen-limited condition. Further, the model can mechanistically explain the overproduction of a drug susceptibility biomarker in the P. aeruginosa mutants. Finally, we use iSD1509 to demonstrate the drug potentiation by metabolite supplementation, and elucidate the mechanisms behind the phenotype, which agree with experimental results.

FoRSR1 Is Important for Conidiation, Fusaric Acid Production, and Pathogenicity in Fusarium oxysporum f. sp. ginseng.

The root rot disease caused by Fusarium oxysporum f. sp. ginseng is one of the most destructive diseases of ginseng, an economically important herb. However, little is known about the pathogen's toxin biosynthesis or the molecular mechanisms regulating infection of ginseng. In this study we identified and functionally characterized the FoRSR1 gene that encodes a Ras-related (RSR) small GTPase homologous to yeast Rsr1 in F. oxysporum f. sp. ginseng. Disruption of FoRSR1 resulted in a significant reduction in mycelial dry weight in liquid cultures, although vegetative growth rate was not affected on culture plates. Notably, the Forsr1 mutant exhibited blunted and swollen hyphae with multi-nucleated compartments. It produced fewer and morphologically abnormal conidia and was defective in chlamydospore formation. In infection assays with ginseng roots, the Forsr1 mutant was significantly less virulent and caused only limited necrosis at the wounding sites. Deletion of FoRSR1 also affected pigmentation, autophagy, and production of fusaric acid. Furthermore, the expression of many candidate genes involved in secondary metabolism was significantly downregulated in the mutant, suggesting that FoRSR1 is also important for secondary metabolism. Overall, our results indicated that FoRSR1 plays important roles in conidiation, vacuolar morphology, secondary metabolism, and pathogenesis in F. oxysporum f. sp. ginseng.

A Nitrate-Sensing Domain-Containing Chemoreceptor Is Required for Successful Entry and Virulence of Dickeya dadantii 3937 in Potato Plants.

Nitrate metabolism plays an important role in bacterial physiology. During the interaction of plant-pathogenic bacteria with their hosts, bacteria face variable conditions with respect to nitrate availability. Perception mechanisms through the chemosensory pathway drive the entry and control the colonization of the plant host in phytopathogenic bacteria. In this work, the identification and characterization of the nitrate- and nitrite-sensing (NIT) domain-containing chemoreceptor of Dickeya dadantii 3937 (Dd3937) allowed us to unveil the key role of nitrate sensing not only for the entry into the plant apoplast through wounds but also for infection success. We determined the specificity of this chemoreceptor to bind nitrate and nitrite, with a slight ligand preference for nitrate. Gene expression analysis showed that nitrate perception controls not only the expression of nitrate reductase genes involved in respiratory and assimilatory metabolic processes but also the expression of gyrA, hrpN, and bgxA, three well-known virulence determinants in Dd3937.

The leucine-responsive regulatory protein SCAB_Lrp modulates thaxtomin biosynthesis, pathogenicity, and morphological development in Streptomyces scabies.

Streptomyces scabies is the best-characterized plant-pathogenic streptomycete, which is a special species among the large genus Streptomyces. The pathogenicity of S. scabies relies on the production of the secondary metabolite thaxtomin A. Little is known about the molecular mechanisms underlying the regulation of thaxtomin biosynthesis in S. scabies beyond the pathway-specific activator TxtR and the cellulose utilization repressor CebR. The leucine-responsive regulatory protein (Lrp) family modulates secondary metabolism in nonpathogenic streptomycetes. However, the regulatory relationship between the Lrp and pathogenic streptomycetes remains unknown. In this study, we demonstrated that SCAB_Lrp (SCAB_77931) from S. scabies significantly affects thaxtomin biosynthesis, pathogenicity, and morphological development. SCAB_Lrp deletion resulted in a dramatic decline in thaxtomin A production and a low-virulence phenotype of S. scabies. An in-depth dissection of the regulatory mechanism of SCAB_Lrp revealed that it positively regulates the transcription of the thaxtomin biosynthetic gene cluster by directly binding to the promoter of the cluster-situated regulator gene txtR. SCAB_Lrp also controls the morphological development of S. scabies by directly activating the transcription of amfC, whiB, and ssgB. SCAB_Lrp directly controls the transcription of its own gene by binding a specific sequence (5'-GGACAGTCGCCGTGCTACG-3'). Moreover, phenylalanine and methionine have been characterized as SCAB_Lrp effectors by strengthening the binding affinity and complex status between SCAB_Lrp and DNA. Our findings characterize a multifunctional regulatory protein, SCAB_Lrp, that controls secondary metabolism, pathogenicity, and sporulation in S. scabies and provide new insights into the complex regulatory network that modulates thaxtomin phytotoxins in pathogenic Streptomyces.

A Novel Biosynthetic Gene Cluster Across the Pantoea Species Complex Is Important for Pathogenicity in Onion.

Onion center rot is caused by at least four species of genus Pantoea (P. ananatis, P. agglomerans, P. allii, and P. stewartii subsp. indologenes). Critical onion pathogenicity determinants for P. ananatis were recently described, but whether those determinants are common among other onion-pathogenic Pantoea species remains unknown. In this work, we report onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic gene clusters present separately in different strains of onion-pathogenic P. stewartii subsp. indologenes. One cluster is similar to the previously described HiVir phosphonate biosynthetic cluster identified in P. ananatis and another is a novel putative phosphonate biosynthetic gene cluster, which we named Halophos. The Halophos gene cluster was also identified in P. allii strains. Both clusters are predicted to be phosphonate biosynthetic clusters based on the presence of a characteristic phosphoenolpyruvate phosphomutase (pepM) gene. The deletion of the pepM gene from either HiVir or Halophos clusters in P. stewartii subsp. indologenes caused loss of necrosis on onion leaves and red onion scales and resulted in significantly lower bacterial populations compared with the corresponding wild-type and complemented strains. Seven (halB to halH) of 11 genes (halA to halK) in the Halophos gene cluster are required for onion necrosis phenotypes. The onion nonpathogenic strain PNA15-2 (P. stewartii subsp. indologenes) gained the capacity to cause foliar necrosis on onion via exogenous expression of a minimal seven-gene Halophos cluster (genes halB to halH). Furthermore, cell-free culture filtrates of PNA14-12 expressing the intact Halophos gene cluster caused necrosis on onion leaves consistent with the presence of a secreted toxin. Based on the similarity of proteins to those with experimentally determined functions, we are able to predict most of the steps in Halophos biosynthesis. Together, these observations indicate that production of the toxin phosphonate seems sufficient to account for virulence of a variety of different Pantoea strains, although strains differ in possessing a single but distinct phosphonate biosynthetic cluster. Overall, this is the first report of onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Complementary Regulation of BfmRS Two-Component and AbaIR Quorum Sensing Systems to Express Virulence-Associated Genes in Acinetobacter baumannii.

Acinetobacter baumannii expresses various virulence factors to adapt to hostile environments and infect susceptible hosts. This study investigated the regulatory network of the BfmRS two-component and AbaIR quorum sensing (QS) systems in the expression of virulence-associated genes in A. baumannii ATCC 17978. The ΔbfmS mutant exhibited a significant decrease in surface motility, which presumably resulted from the low expression of pilT and A1S_0112-A1S_0119 gene cluster. The ΔbfmR mutant displayed a significant reduction in biofilm and pellicle formation due to the low expression of csu operon. The deletion of abaR did not affect the expression of bfmR or bfmS. However, the expression of abaR and abaI was upregulated in the ΔbfmR mutant. The ΔbfmR mutant also produced more autoinducers than did the wild-type strain, suggesting that BfmR negatively regulates the AbaIR QS system. The ΔbfmS mutant exhibited no autoinducer production in the bioassay system. The expression of the A1S_0112-A1S_0119 gene cluster was downregulated in the ΔabaR mutant, whereas the expression of csu operon was upregulated in this mutant with a high cell density. In conclusion, for the first time, we demonstrated that the BfmRS-AbaIR QS system axis regulated the expression of virulence-associated genes in A. baumannii. This study provides new insights into the complex network system involved in the regulation of virulence-associated genes underlying the pathogenicity of A. baumannii.

Molecular investigation of prevalence, phenotypic and genotypic diversity, antibiotic resistance, frequency of virulence genes and genome sequencing in Pseudomonas aeruginosa strains isolated from lobster.

AIM: Aquatic organisms are too susceptible to the increased growth of bacterial contamination. It seems that preventive measures should be prioritized to reduce bacterial load, and improve the health situation of marine-based product consumers. Hence, this study is aimed at molecular investigation of the prevalence of Pseudomonas aeruginosa as one of the most food-borne pathogens, antibiotic resistance, and virulence factor encoding gens in lobster samples. METHODOLOGY: After the collection of aquatic samples from Isfahan and Chabahar city during the summer and autumn seasons, they were cultured, and confirmed by biochemistry tests. Then, they were investigated for antibiotic resistance by the Kirby Bauer method. Then, antibiotic resistance, virulence factor encoding genes, and Multi-Drug Resistance (MDR) patterns were analyzed. Statistical analysis was done by SPSS through chi-square tests. RESULTS: Bacterial contamination in samples taken from Isfahan city was higher than in Chabahar city despite having a cooler climate on summer days. Antibiotic resistance to piperacillin in fresh shrimp samples taken in summer In Isfahan city was contrary to its usage as a front-line antibiotic agent for Pseudomonas aeruginosa. Lowered MDR pattern in frozen samples, was related to the varied expression of antibiotic resistance, highlighting the importance of regulations for cold chain in storage, transportation, and distribution of marine samples, especially when compared to fresh shrimps. CONCLUSION: Food-borne pathogens, antibiotic resistance, and their virulence factors are of clinical and environmental importance. Results of our study indicated a high rate of frequency for Pseudomonas aeruginosa isolated from marine samples, antibiotic resistance, antibiotic resistance encoding genes, virulence factors encoding genes, and MDR. Maintenance of the cold chain, and proper food processing, have indispensable roles in the preservation, and reduction of Pseudomonas aeruginosa frequency in aquatic organisms.

Protease Inhibitors from Solanum chacoense Inhibit Pectobacterium Virulence by Reducing Bacterial Protease Activity and Motility.

Potato is a major staple crop, and necrotrophic bacterial pathogens such as Pectobacterium spp. are a major threat to global food security. Most lines of cultivated potato (Solanum tuberosum) are susceptible to Pectobacterium spp., but some lines of wild potato are resistant, including Solanum chacoense M6. Despite the discovery of resistance in wild potatoes, specific resistance genes are yet to be discovered. Crude protein extract from M6 had a global effect on Pectobacterium brasiliense Pb1692 (Pb1692) virulence phenotypes. Specifically, M6 protein extracts resulted in reduced Pectobacterium exo-protease activity and motility, induced cell elongation, and affected bacterial virulence and metabolic gene expression. These effects were not observed from protein extracts of susceptible potato S. tuberosum DM1. A proteomics approach identified protease inhibitors (PIs) as candidates for S. chacoense resistance, and genomic analysis showed higher abundance and diversity of PIs in M6 than in DM1. We cloned five PIs that are unique or had high abundance in M6 compared with DM1 and purified the proteins (g18987, g28531, g39249, g40384, g6571). Four of the PIs significantly reduced bacterial protease activity, with strongest effects from g28531 and g6571. Three PIs (g18987, g28531, g6571) inhibited disease when co-inoculated with Pectobacterium pathogens into potato tubers. Two PIs (g28531, g6571) also significantly reduced Pb1692 motility and are promising as resistance genes. These results show that S. chacoense PIs contribute to bacterial disease resistance by inhibiting exo-proteases, motility, and tuber maceration and by modulating cell morphology and metabolism. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative analysis of virus pathogenicity and resistance-breaking between the P- and A-type from the beet necrotic yellow vein virus using infectious cDNA clones.

The A-type of beet necrotic yellow vein virus (BNYVV) is widely distributed in Europe and is one of the major virus types causing rhizomania disease in sugar beet. The closely related P-type is mainly limited to a small region in France (Pithiviers). Both virus types possess four RNAs (RNA1-4), but the P-type harbours an additional fifth RNA species (RNA5). The P-type is associated with stronger disease symptoms and resistance-breaking of Rz1, one of the two resistance genes which are used to control BNYVV infection. These characteristics are presumably due to the presence of RNA5, but experimental evidence for this is lacking. We generated the first infectious cDNA clone of BNYVV P-type to study its pathogenicity in sugar beet in comparison to a previously developed A-type clone. Using this tool, we confirmed the pathogenicity of the P-type clone in the experimental host Nicotiana benthamiana and two Beta species, B. macrocarpa and B. vulgaris. Independent of RNA5, both the A- and the P-type accumulated in lateral roots and reduced the taproot weight of a susceptible sugar beet genotype to a similar extent. In contrast, only the P-type clone was able to accumulate a virus titre in an Rz1-resistant variety whereas the A-type clone failed to infect this variety. The efficiency of the P-type to overcome Rz1 resistance was strongly associated with the presence of RNA5. Only a double resistant variety, harbouring Rz1 and Rz2, prevented an infection with the P-type. Reassortment experiments between the P- and A-type clones demonstrated that both virus types can exchange whole RNA components without losing the ability to replicate and to move systemically in sugar beet. Although our study highlights the close evolutionary relationship between the two virus types, we were able to demonstrate distinct pathogenicity properties that are attributed to the presence of RNA5 in the P-type.

Photo-oxidative stress response and virulence traits are co-regulated in E. faecalis after antimicrobial photodynamic therapy.

Knowledge of photo-oxidative stress responses in bacteria that survive antimicrobial photodynamic therapy (aPDT) is scarce. Whereas aPDT is attracting growing clinical interest, subsequent stress responses are crucial to evaluate as they may lead to the up-regulation of pathogenic traits. Here, we aimed to assess transcriptional responses to sublethal aPDT-stress and identify potential connections with virulence-related genes. Six Enterococcus faecalis strains were investigated; ATCC 29212, three dental root-canal isolates labelled UmID1, UmID2 and UmID3 and two vancomycin-resistant isolates labelled A1 and A2. TMPyP was employed as a photosensitiser. A viability dose-response curve to increasing concentrations of TMPyP was determined by culture plating. Differential expression of genes involved in oxidative stress responses (dps and hypR), general stress responses (dnaK, sigma-factorV and relA), virulence-related genes (ace, fsrC and gelE) and vancomycin-resistance (vanA) was assessed by reverse-transcription qPCR. TMPyP-mediated aPDT inactivated all strains with comparable efficiencies. TMPyP at 0.015 µM was selected to induce sublethal photo-oxidative stress. Despite heterogeneities in gene expression between strains, transcriptional profiles revealed up-regulations of transcripts dps, hypR as well as dnaK and sigma factorV after exposure to TMPyP alone and to light-irradiated TMPyP. Specifically, the alternative sigma factorV reached up to 39 ± 113-fold (median ± IQR) (p = 0.0369) in strain A2. Up-regulation of the quorum sensing operon, fsr, and its downstream virulence-related gelatinase gelE were also observed in strains ATCC-29212, A1, A2 and UmID3. Finally, photo-oxidative stress induced vanA-type vancomycin-resistance gene in both carrier isolates, reaching up to 3.3 ± 17-fold in strain A2 (p = 0.015). These findings indicate that, while aPDT successfully inactivates vancomycin-resistant and naïve strains of E. faecalis, subpopulations of surviving cells respond by co-ordinately up-regulating a network of genes involved in stress survival and virulence. This includes the induction of vancomycin-resistance genes in carrier isolates. These data may provide the mechanistic basis to circumvent bacterial responses and improve future clinical protocols.

Alhagi maurorum extract modulates quorum sensing genes and biofilm formation in Proteus mirabilis.

Proteus mirabilis (P. mirabilis) is a frequent cause of catheter-associated urinary tract infections. This study aims to investigate the anti-infective effect of Alhagi maurorum extract (AME), the traditional medicinal plant in the middle east, on the biofilm-forming P. mirabilis isolates. Hydroalcoholic extract and oil of A. maurorum were characterized by HPLC and GC-MS. The antiproliferative, anti-biofilm, and bactericidal activity of AME at various concentrations were assessed by turbidity, crystal violet binding, and agar well diffusion assays, respectively. The AME's effect on adhesion and quorum sensing (QS) were investigated by in vitro adhesion assay on cell culture and agar overlay assay using Janthinobacterium lividum (ATCC 12472) as a biosensor strain. In addition, the expression level of selected genes involved in QS and biofilm regulation were determined by quantitative Real-Time PCR. Furthermore, the bladder phantom model was created to evaluate the assays and investigate the catheter's calcium deposition. The most effective chemical compounds found in AME were tamarixetin, quercetin, and trans-anethole. Although AME did not inhibit swarming motility, it reduced biofilm production and exerted a concentration-dependent anti-adhesive and anti-QS activity against P. mirabilis. AME also downregulated the expression level of selected genes involved in biofilm formation and QS. This study showed that AME as a natural compound reduced biofilm formation of P. mirabilis by targeting virulence factor genes, quorum sensing, and other strategies that include preventing the adhesion of P. mirabilis to the cells. The results suggest that A. maurorum extract might have the potential to be considered for preventing UTIs caused by P. mirabilis.