Virulence-related Genes Research Articles

Pangenomic Analysis of Dickeya dianthicola Strains Related to the Outbreak of Blackleg and Soft Rot of Potato in the United States

Dickeya dianthicola has caused an outbreak of blackleg and soft rot of potato in the eastern half of the United States since 2015. To investigate genetic diversity of the pathogen, a comparative analysis was conducted on genomes of D. dianthicola strains. Whole genomes of 16 strains from the United States outbreak were assembled and compared with 16 previously sequenced genomes of D. dianthicola isolated from potato or carnation. Among the 32 strains, eight distinct clades were distinguished based on phylogenomic analysis. The outbreak strains were grouped into three clades, with the majority of the strains in clade I. Clade I strains were unique and homogeneous, suggesting a recent incursion of this strain into potato production from alternative hosts or environmental sources. The pangenome of the 32 strains contained 6,693 genes, 3,377 of which were core genes. By screening primary protein subunits associated with virulence from all U.S. strains, we found that many virulence-related gene clusters, such as plant cell wall degrading enzyme genes, flagellar and chemotaxis related genes, two-component regulatory genes, and type I/II/III secretion system genes, were highly conserved but that type IV and type VI secretion system genes varied. The clade I strains encoded two clusters of type IV secretion systems, whereas the clade II and III strains encoded only one cluster. Clade I and II strains encoded one more VgrG/PAAR spike protein than did clade III. Thus, we predicted that the presence of additional virulence-related genes may have enabled the unique clade I strain to become predominant in the U.S. outbreak.

Site specific incidence rate of virulence related genes of enteroaggregative Escherichia coli and association with enteric inflammation and growth in children

There is a lack of information highlighting the possible association between strain carrying genes of enteroaggregative Escherichia coli (EAEC) and environmental enteric dysfunction (EED) and on linear growth during childhood. Strain carrying genes of EAEC from stool samples collected from 1705 children enrolled in the MAL-ED birth cohort were detected by TaqMan Array Cards. We measured site-specific incidence rate by using Poisson regression models, identified the risk factors and estimated the associations of strain carrying genes of EAEC with the composite EED score and linear growth at 24 months of age. Overall highest incidence rate (43.3%) was found among children having infection with the aggR gene, which was the greatest in Tanzania (56.7%). Low maternal education, lack of improved floor, and ownership of domestic cattle were found to be risk factors for EAEC infection. In the multivariate models, after adjusting the potential covariates, strain carrying genes of EAEC showed strong positive associations with the EED scores and with poor linear growth at 24 months of age. Our analyses may lay the cornerstone for a prospective epidemiologic investigation for a potential vaccine development aimed at reducing the burden of EAEC infections and combat childhood malnutrition.

A thermal adaptation landscape related to virulence in Mucor irregularis transcriptional profiles

Our study aimed to better understand the different thermal adaptation in Mucor irregularis (M.irregularis) strains under high temperature and the involved virulence-related genes, and to offer more appropriate explanation for the diverse pathogenicity of M.irregularis in human infections. M.irregularis isolates were incubated at 30 and 35°C for Illumina HiSeq technology (RNA-seq), as well as the virulence difference detected through Galleria mellonella infection models. We verified their transcriptional profile with RT-PCR and analysed differentially expressed genes with GO and KEGG annotations. All 25 isolates formed the biggest colonies at 28°C and did not grow at 37°C, while were differently inhibited at 22 and 35°C. Six selected M.irregularis displayed virulence in sync with their growth condition at high temperature. From the outcomes of RNA-seq, a total of 1559 differentially expressed genes (FC≥2, FDR<0.05) were obtained, of which 1021 genes were upregulated, and 538 genes were downregulated. Cell wall structure genes related to Ras-like and GH16 proteins, influx-efflux pumps consist of transmembrane proteins as ABC and MFS proteins, and metabolic genes as DGKɛ and Hsfs, seem to be essential in thermal adaptation and virulence of M.irregularis. We found some common genes expressed at high temperature, while some others specifically related to M.irregularis isolates with different virulence and thermal adaptation. Further research of genes involved in the pathogenic process is needed for the development of potential targeted antifungal.

Abf1 Is an Essential Protein That Participates in Cell Cycle Progression and Subtelomeric Silencing in Candida glabrata.

Accurate DNA replication and segregation is key to reproduction and cell viability in all organisms. Autonomously replicating sequence-binding factor 1 (Abf1) is a multifunctional protein that has essential roles in replication, transcription, and regional silencing in the model yeast Saccharomyces cerevisiae. In the opportunistic pathogenic fungus Candida glabrata, which is closely related to S. cerevisiae, these processes are important for survival within the host, for example, the regulation of transcription of virulence-related genes like those involved in adherence. Here, we describe that CgABF1 is an essential gene required for cell viability and silencing near the telomeres, where many adhesin-encoding genes reside. CgAbf1 mediated subtelomeric silencing depends on the 43 C-terminal amino acids. We also found that abnormal expression, depletion, or overexpression of Abf1, results in defects in nuclear morphology, nuclear segregation, and transit through the cell cycle. In the absence of ABF1, cells are arrested in G2 but start cycling again after 9 h, coinciding with the loss of cell viability and the appearance of cells with higher DNA content. Overexpression of CgABF1 causes defects in nuclear segregation and cell cycle progression. We suggest that these effects could be due to the deregulation of DNA replication.

Epidemiological characteristics of Streptococcus agalactiae in tilapia in China from 2006 to 2020

Group B Streptococcus (GBS) is the major causative pathogen of streptococcosis in tilapia, leading to a massive economic loss in the aquaculture industry worldwide. To date, the epidemiological characterization of tilapia GBS in China remains limited. Thus, the aim of this study was to determine the phenotype and genotype of GBS strains in tilapia in China from 2006 to 2020. A total of 678 GBS strains were isolated from farmed tilapia in the past decade. The CAMP reaction of GBS strains was investigated, and the cfb gene of the CAMP negative strains was sequenced and analysed. The genotypic distribution of these strains was determined by molecular serotyping, multi-locus sequence typing (MLST), virulence genotyping, and prophage genotyping. All GBS strains were divided into two serotypes (Ia and Ib), two sequence types (STs; ST7 and ST261), five virulence genotypes (V1–V5), and three prophage genotypes (P1-P3). A total of six genotypes (I–VI) were obtained based on serotyping, MLST, virulence genotyping, and prophage genotyping. Among them, strains in genotypes I, II, III, and IV were primarily prevalent from 2006 to 2015, and those in genotypes V and VI were the dominant epidemic strains from 2012 to 2020. Moreover, genotype VI strains showed a substantial population explosion from 2011 to 2020 and were mainly distributed in the geographically proximate provinces of Guangdong, Guangxi, Hainan, and Fujian. This is first report of tilapia Ia-GBS CAMP negative strains, and the strains were probably from the same hatchery. Genetic diversity in tilapia GBS strains has been high in China over the past decade, especially from 2010 to 2015. The genotypes of the tilapia GBS strains underwent obvious variation, and the prevalent strains in recent years have harboured more virulence-related genes. This study provides a better understanding of the epidemiology, pathogenesis, and evolutionary mechanism of tilapia GBS strains.

Genomic characterization and pathogenicity analysis of the Vibrio mimicus Y4 causing red body disease in Macrobrachium nipponense

Vibrio mimicus distributes widely in the aquatic ecosystem and causes vibriosis in various aquatic animals. For the past few years, Macrobrachium nipponense has been suffering from red body disease with significant economic loss in Jiangsu, and V. mimicus is recognized to be the etiological agent of the disease. In this study we investigated the pathogenicity and antibiotic resistance of V. mimicus Y4 which caused red body disease in M. nipponense in the genomic level. In addition, the high pathogenicity of V. mimicus Y4 was further verified by survival analysis and lesions of hepatopancreas and gill tissues of M. nipponense. The complete genome sequence analysis showed that its genome contained two circular chromosomes and one plasmid with a total size of 4,347,065 bp, which harbored 4084 coding genes. Among these genes, 387 genes were predicted as virulence related genes which could be classified as type IV pilus, flagella, hemolysin, heme receptors, type VI secretion system, and so on, which are involved in the colonization, invasion and destruction of host tissues; 129 genes were predicted as antibiotic resistance genes, including aminoglycoside resistant, macrolide resistant, beta-lactam resistant, and polymyxin resistant genes, which agree with the antibiotic resistance results, which indicated V. mimicus Y4 to be a multidrug-resistant bacterium. The LD50 of the strain Y4 to M. nipponense was 3.28 × 105 CFU/mL at 96 h post-infection. Histopathological analysis exhibited obvious severe lesions of hepatopancreas and gill tissues of diseased M. nipponense, such as the basal laminae of the hepatopancreatic tubules were rupture, vacuolation were decreased, and gill filament cells were broken in diseased prawns. This study provides a theoretical basis for better understanding of the pathogenicity and antibiotic resistance, which might contribute to the prevention of V. mimicus in the future.

Antimicrobial Resistance and Genomic Characterization of Six New Sequence Types in Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Pakistan.

Pseudomonas aeruginosa (P. aeruginosa) is a major bacterial pathogen associated with a variety of infections with high mortality rates. Most of the clinical P. aeruginosa isolates belong to a limited number of genetic subgroups characterized by multiple housekeeping genes’ sequences (usually 5–7) through the Multi-Locus Sequence Typing (MLST) scheme. The emergence and dissemination of novel multidrug-resistant (MDR) sequence types (ST) in P. aeruginosa pose serious clinical concerns. We performed whole-genome sequencing on a cohort (n = 160) of MDR P. aeruginosa isolates collected from a tertiary care hospital lab in Pakistan and found six isolates belonging to six unique MLST allelic profiles. The genomes were submitted to the PubMLST database and new ST numbers (ST3493, ST3494, ST3472, ST3489, ST3491, and ST3492) were assigned to the respective allele combinations. MLST and core-genome-based phylogenetic analysis confirmed the divergence of these isolates and positioned them in separate branches. Analysis of the resistome of the new STs isolates revealed the presence of genes blaOXA-50, blaPAO, blaPDC, blaVIM-2, aph(3′)-IIb, aac(6′)-II, aac(3)-Id, fosA, catB7, dfrB2, crpP, merP and a number of missense and frame-shift mutations in chromosomal genes conferring resistance to various antipseudomonal antibiotics. The exoS, exoT, pvdE, rhlI, rhlR, lasA, lasB, lasI, and lasR genes were the most prevalent virulence-related genes among the new ST isolates. The different genotypic features revealed the adaptation of these new clones to a variety of infections by various mutations in genes affecting antimicrobial resistance, quorum sensing and biofilm formation. Close monitoring of these antibiotic-resistant pathogens and surveillance mechanisms needs to be adopted to reduce their spread to the healthcare facilities of Pakistan. We believe that these strains can be used as reference strains for future comparative analysis of isolates belonging to the same STs.

GENOTYPIC AND PHENOTYPIC CHARACTERIZATION OF BACTERIOCINOGENIC LACTIC ACID BACTERIAL STRAINS FOR POSSIBLE BENEFICIAL, VIRULENCE, AND ANTIBIOTIC RESISTANCE TRAITS

Lactic acid bacteria (LAB) are a diverse group of microorganisms that can be found in natural habitats and are used in a wide range of industries, especially in the food sector. They are regarded Generally Recognized as Safe and are commonly used as a starter culture, probiotics, and biopreservative. However, some LAB genera are excluded from the Qualified Presumption of Safety (QPS) list due to the possibility that they have virulence and antibiotic resistance genetic determinants. The objective of this study was to investigate the beneficial, virulence, and antibiotic resistance-related genes in 10 well-characterized bacteriocinogenic LAB strains. The auto-aggregation, co-aggregation, cell surface hydrophobicity, and bile salt tolerance were also evaluated to examine their potential as bacteriocinogenic probiotics and/or starter culture. Results showed that all tested strains showed abilities for auto-aggregation at 4oC and 37oC, co-aggregation with S. aureus JCM8704, S. typhimurium BIOTECH1826, and E. coli DH5α, and significant cell-surface hydrophobicity. However, only a few strains were able to withstand the media treated with 0.3% bile salt. Among the tested bacteriocinogenic LAB strains, L. lactis IO-1 and C. divergens V41 had the maximum values for auto-aggregation at 4oC and 37oC, respectively. C. divergens V41 also exhibited the highest percentage for cell surface hydrophobicity. E. faecium NKR-5-3 showed the highest co-aggregation percentages with all indicator strains. Our findings also showed that the tested isolates presented distinct combinations of virulence-related genes. Only two of ten bacteriocinogenic LAB strains exhibited presence of multiple virulence genes. Lactococcus sp. QU12 was found to have a high frequency of beneficial and virulence genes, with 2 out of 7 genes present encoding beneficial factor and 11 out of 13 genes encoding virulence factor. Lactococcus sp. QU12 and L. lactis IO-1 were also positive for tetracycline resistance gene tetM and aminoglycoside resistance gene aphA-2, respectively, and transposon genes. Moreover, only a few LAB isolates tested positive for 2 out of 8 antibiotic resistance classes. Although few, the substantial danger of these genes being transferred and acquired cannot be overlooked as this could potentially cause serious health concerns in the future. These results suggest that despite the promising properties of bacteriocinogenic LAB, careful safety evaluation of these strains should be a prerequisite before using these in food systems.

Molecular Characterization Based on Whole-Genome Sequencing of Streptococcus pneumoniae in Children Living in Southwest China During 2017-2019.

BackgroundStreptococcus pneumoniae is an important pathogen causing high morbidity and high mortality in children and undergoes frequent recombination for capsule switching to neutralize the 13-valent pneumococcal conjugate vaccine (PCV13). This study aimed to investigate the prevalence, and molecular characteristics including serotypes and antibiotic susceptibility of S. pneumoniae isolated from children living in Southwest China from 2017 to 2019 to facilitate the selection of effective vaccine formulations and appropriate antibiotic treatment regimens.MethodsThis study was conducted at West China Second University Hospital (Chengdu, Sichuan Province, China), Zunyi Medical University Third Affiliated Hospital/First People’s Hospital of Zunyi (Zunyi, Guizhou Province, China) and Chengdu Jinjiang District Maternal and Child Healthcare Hospital (Chengdu, Sichuan Province, China). Demographic and clinical characteristics of children infected with S. pneumoniae were collected and analysed. Next-generation sequencing and sequence analysis were used to determine the serotypes, sequence types, antibiotic resistance and potential protein vaccine target genes of the pneumococcal isolates. The coverage rate provided by PCV13 was estimated by calculating the percentage of the specific serotypes that were specifically the PCV13-included serotypes. Antimicrobial susceptibility was determined by the microdilution broth method.ResultsThe most prevalent pneumococcal serotypes were 19F (25.8%), 19A (14.1%), 6B (12.5%), 6A (9.4%) and 14 (7.8%). The predominant STs were ST271 (23.3%), ST320 (15.5%) and ST90 (8.6%), dominated by the clonal complex Taiwan19F-14 (39.1%). The coverage rate of PCV13 was 77.3% in all the isolates, with relatively higher values in invasive isolates (86.4%). Over the decade, the rates of resistance to penicillin, amoxicillin and cefotaxime were 5.6%, 5.3% and 5.1%, respectively, with significantly higher values in invasive isolates (22.4%, 14.9% and 11.9%). Almost all the isolates were resistant to erythromycin (99.1%) and clindamycin (95.9%). All isolates carried virulence-related genes, including ply, psaA, piaA, piuA, phtE, nanA, pepO, danJ, pvaA, clpP, pcsB, stkP, potD, and strH. The carriage of virulence and resistance genes varied among serotypes and clades, with serotype 19F/ST271 showing higher resistance to antibiotics and being more likely to carry pilus genes and other virulence genes.ConclusionThese data provide valuable information for the understanding of pneumococcal pathogenesis, antimicrobial resistance and the development of protein-based vaccines against pneumococcal infection.

Characterization of Faecal Enterococci from Wild Birds in Turkey and Its Importance in Antimicrobial Resistance

This research aimed to investigate the diversity of faecal enterococci isolated from wild birds, to detecttheir antibiotic resistance patterns and to determine their distribution of genes related to vancomycin resistance. Additionally, to investigate their virulence factors that are important in the development of the disease. One hundred seven cloacal/rectal samples were inoculated onto Enterococcus Agar, and presumptive colonies were identified and confirmed by PCR. Multiplex PCR assays were used to screen vanA, vanB, vanC1 and vanC2/3. The virulence-related genes; ace, gelE, efa and agg were determined by PCR. Among the 103 enterococci, 62 E.faecalis, 23 E.faecium 3 E.gallinarum, 2 E.durans, 1 E.casseliflavus and 12 Enterococcus spp. were identified. Of the 103 enterococci, 26 were found to be resistant against to three or more antibiotics. The highest percentages were detected for chloramphenicol (52%), tetracycline (33%) and erythromycin (30%). Two E.gallinarum isolates were harboring three virulence factors, and one isolate was carrying a single virulence factor. There is no virulence factor in the E.casseliflavus isolate. Also, vanA and vanB genes were not found. Forty-two of 103 enterococci were harboring virulence factors, more frequently in E.faecalis. Forty-two enterococci carried efa A, 31 isolates carried gel E, and ace was found in 18 isolates. Virulence gene agg was not detected. When the results of the study were evaluated in general, multiple drug resistance was described as 25%. Considering the risk of polluting the water resources of wild animals, it is suggested that the continuity of this type of epidemiological study in wildlife animals is necessary. In conclusion, the wild birds may act as substantial reservoirs carrying antimicrobial resistance among enterococci and estimate the potential risk for man, pets and farm animals.

The antiviral drug efavirenz reduces biofilm formation and hemolysis by Staphylococcus aureus

Introduction. Biofilm formation and hemolysis are closely related to the pathogenicity of Staphylococcus aureus.Hypothesis/Gap Statement. Strategies that reduce the mortality of S. aureus infections may involve novel antimicrobials and/or drugs that decrease S. aureus virulence, such as biofilm formation. The antiviral drug efavirenz is a non-nucleoside reverse transcriptase inhibitor, which also has shown antibacterial effect on Bacillus subtilis and Escherichia coli. Its effect on pathogen virulence has not yet been explored.Aim. This study investigates the antimicrobial and anti-virulence effect of efavirenz on S. aureus.Methodology. Biofilm biomasses were detected by crystal violet staining. Hemolysis activities of S. aureus were determined by rabbit erythrocytes lysis assay. RNA levels of transcriptional regulatory genes, biofilm-related genes, and virulence-related genes of S. aureus were determined by RT-qPCR.Results. Efavirenz showed an inhibitory effect on the growth of S. aureus, Enterococcus faecalis and Streptococcus agalactiae at 50 µM. Efavirenz significantly inhibited biofilm formation of both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) at 25 µM, but did not affect the growth of planktonic S. aureus cells. Moreover, hemolysis by S. aureus was inhibited by efavirenz at 25 µM. The expression levels of RNA transcriptional regulatory genes (agrA, agrC, sigB, saeR and saeS), biofilm-related genes (cidA, clfA, clfB, fnbA, fnbB), and virulence-related genes (hla, hld, staphopain B, alpha-3 PSM, beta PSM, delta PSM) of S. aureus decreased significantly at 25 µM efavirenz.Conclusion. Efavirenz inhibits S. aureus biofilm formation and virulence in vitro.

Extracellular Vesicles: Emerging Players in Plant Defense Against Pathogens.

Communication between plants and interacting microorganisms requires functional molecule trafficking, which is essential for host defense and pathogen virulence. Extracellular vesicles (EVs) are single membrane-bound spheres that carry complex cargos, including lipids, proteins, and nucleic acids. They mediate cell-to-cell communication via the transfer of molecules between cells. Plant EVs have been isolated from many plant species and play a prominent role in immune system modulation and plant defense response. Recent studies have shown that plant EVs are emerging players in cross-kingdom regulation and contribute to plant immunity by mediating the trafficking of regulatory small RNA into pathogens, leading to the silencing of pathogen virulence-related genes. This review summarizes the current understanding of plant EV isolation technologies, the role of plant EVs in plant immunity, and the mechanism of plant EV biogenesis, as well as approaches for how these findings can be developed into innovative strategies for crop protection.

Effects of osmotic and high pressure stress on expression of virulence factors among Enterococcus spp. isolated from food of animal origin

The aim of study was to determine the occurrence of virulence factors and virulence-related genes among enterococci isolated from food of animal origin and effects of osmotic and high pressure stress on expression of virulence-related genes. The number of 78 isolates were analyzed. None of them showed a strong ability to form biofilm, 38.5% (n = 30) had the slime production ability, 41% (n = 32) had gelatinase activity, γ -type hemolysis was observed in 55% of isolates, and α-type hemolysis in 45%. All of the isolates carried 1–13 virulence-related genes. The most common genes were gelE (85.9%), sprE (78.2%) and asa1 (75.6%). There were also observed changes in the expression of the gelE, esp, asa1 and cylL genes in response to various NaCl concentration and high pressure processing. Results obtained in this study indicate that enterococci isolated from food may act as reservoirs of virulence genes. The presence of virulence factors among enterococci, especially the ability to biofilm formation is important for food safety and the protection of public health. The results presented in our work demonstrate that stress that can occur during food preservation and food processing can induce the changes in the virulence-related genes expression.

Characterization of Emerging Pathogens Carrying blaKPC-2 Gene in IncP-6 Plasmids Isolated From Urban Sewage in Argentina.

Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we isolated a KPC-2-producing ST2787 Klebsiella quasipneumoniae subsp. quasipneumoniae (WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all β-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly, bla KPC-2 was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel non-typable/IncP-6 hybrid plasmid in a newly assigned ST1407 Enterobacter asburiae (WW19C) also harboring bla KPC-2. Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (~11 kb) in both plasmids contained a Tn3 transposon disrupted by a Tn3-ISApu-flanked element and the core sequence was composed by ΔISKpn6/bla KPC-2/Δbla TEM-1/ISKpn27. Both strains also carried genes conferring resistance to heavy metals (e.g., arsenic, mercury, lead, cadmium, copper), pesticides (e.g., glyphosate), disinfectants, and several virulence-related genes, posing a potential pathogenic risk in the case of infections. This is the first study documenting bla KPC-2 associated with IncP-6 plasmids in K. quasipneumoniae and Enterobacter cloacae complex from wastewater in Argentina and highlights the circulation of IncP-6 plasmids as potential reservoirs of bla KPC-2 in the environment.

An Integrated Analysis of Intracellular Metabolites and Virulence Gene Expression during Biofilm Development of a Clinical Isolate of Candida tropicalis on Distinct Surfaces.

Emergence of Candida tropicalis, which causes potential life-threatening invasive candidiasis, is often associated with colonization of medical devices as biofilm. Biofilm plays an important role in the virulence of the pathogen because of its complex structure, which provides resistance to conventional antimicrobials. In this study, the metabolic response of a clinical strain of C. tropicalis colonizing three distinct surfaces (polytetrafluoroethylene (PTFE), polystyrene, and polycarbonate) as well as the expression of virulence and stress related genes (ALS3, Hsp21, SAP1, SAP2, SAP3, and CYR1), were explored. Our results showed that lesser biofilm was developed on PTFE compared to polystyrene and polycarbonate. GS-MS metabolic analysis identified a total of 36 metabolites in the intracellular extract of cells grown on polystyrene, polycarbonate, and PTFE, essentially belonging to central carbon metabolism, amino acids, and lipids metabolism. The metabolic analysis showed that saturated and unsaturated fatty acids are preferentially produced during biofilm development on polycarbonate, whereas trehalose and vitamin B6, known as cellular protectors against a variety of stressors, were characteristic of biofilm on PTFE. The results of the transcriptomic analysis consider the different degrees of colonization of the three substrates, being CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA, downregulated in PTFE biofilm compared to polycarbonate or polystyrene biofilms, while Hsp21 was upregulated in concomitance with the potential unfavorable conditions for biofilm formation on PTFE. Overall, this work provides new insights into the knowledge of C. tropicalis biofilm development on surfaces of medical relevance in the perspective of improving the management of Candida infections.

Antibiofilm activities of fatty acids including myristoleic acid against Cutibacterium acnes via reduced cell hydrophobicity

BackgroundCutibacterium acnes is a major colonizer and inhabitant of human skin and contributes to the pathogenesis of acne vulgaris. C. acnes either alone or with Staphylococcus aureus, which also inhabits skin, readily forms biofilms that are often tolerant of conventional antibiotics and the host immune system. It was hypothesized that the amphiphilic nature of some fatty acids (FAs) inhibit C. acnes or mixed biofilm formation. PurposeThe antibacterial and antibiofilm activities of 24 saturated and unsaturated FAs were investigated against C. acnes as well as a mixture of the bacteria C. acnes and S. aureus. MethodsAnti-biofilm assays, antimicrobial assays, confocal laser scanning microscopy, scanning electron microscopy, extracellular polymeric substance production, and microbial adherence to hydrocarbon assay were utilized to elucidate how active FAs influence biofilm development. ResultsSeventeen FAs at 20 µg/ml inhibited C. acnes biofilm formation by 60–99%. The minimum inhibitory concentrations (MICs) of 20 FAs were ≥ 500 µg/ml but 4 medium-chain FAs had MICs in a range 15 to 200 µg/ml. Interestingly, myristoleic acid inhibited biofilm formation at 1 μg/ml. Myristoleic acid also inhibited the formation of S. aureus and mixed C. acnes/S. aureus biofilms. FAs reduced C. acnes hydrophobicity and we found this was generally correlated with their antibiofilm forming efficacies. Transcriptional analyses showed that myristoleic acid modulates the expression of several biofilm-related genes such as lipase, hyaluronate lyase, and virulence-related genes. ConclusionThis study shows myristoleic acid and other FAs inhibit biofilm formation by C. acnes and mixed biofilm formation by C. acnes and S. aureus. Hence, myristoleic acid might be useful for treating or preventing acne and C. acnes associated diseases.

DNA Methyltransferases Regulate Pathogenicity of Botrytis cinerea to Horticultural Crops.

Botrytis cinerea is one of the most destructive fungal pathogens that cause gray mold rot in horticultural products, including fresh fruits, vegetables, and flowers, leading to serious economic losses. B. cinerea is difficult to control because it has strong stress resistance and complex infection modes. The pathogenic mechanisms of B. cinerea have been revealed at multiple levels, but little is known at the epigenetic level. In this study, we first revealed the important role of DNA methyltransferases in regulating the development and pathogenicity of B. cinerea. We showed that two DNA methyltransferases, BcDIM2 and BcRID2, showed a strong synergistic effect in regulating the pathogenicity of B. cinerea. The double knockout mutant ΔBcdim2rid2 showed slower mycelial growth, lower spore germination, attenuated oxidative tolerance, and complete pathogenicity loss on various hosts, which is related to the reduced expression of virulence-related genes in ΔBcdim2rid2 and the induced resistance of the host. Although B. cinerea has multiple DNA methyltransferases, the global methylation level is very low, and few 5mC sites can be detected by BS-seq. These results first revealed the important role and the action mode of DNA methyltransferases in B. cinerea.

Antimicrobial Resistance and Virulence Factor of Streptococcus dysgalactiae Isolated from Clinical Bovine Mastitis Cases in Northwest China.

ObjectiveStreptococcus dysgalactiae is a major pathogen in bovine mastitis. The purpose of this study was to survey the prevalence, antimicrobial resistance, as well as the spread of resistance and virulence-associated gene of S. dysgalactiae.MethodsA total of 60 S. dysgalactiae strains were obtained from 830 milk samples from Holstein cows with clinical mastitis. Antimicrobial resistance was examined by the disk diffusion method. Antimicrobial resistance and virulence genes were investigated by PCR, agarose gel electrophoresis and 16S rRNA gene sequencing.ResultsAll isolates were resistant to tetracycline and showed a high level of resistance to aminoglycoside antibiotics, where 81.67% of the strains were multi-resistant to these ten sorts of antibiotics. In addition, the most prevalent resistance gene in S. dysgalactiae was aphA-1 (98.33%), followed by blaTEM (96.67%), ermB (83.3%), aadA1/aadA2 (78.33%) and tetL (73.33%). Totally, seven virulence genes with 25 combination patterns were detected in these isolates, and each isolates harbored at least one virulence gene. 21.67% of the isolates carried three or more virulence genes, while one strain with seven virulence-related genes and belonged to cfb+lmb+eno+napr+bca+scpB+cyl.ConclusionThese findings indicate that S. dysgalactiae isolated from clinical bovine mastitis cases in Northwest China show a variety of molecular ecology and are highly resistant to antibiotics commonly used in dairy farms. This research will help investigators better understand the pathophysiology S. dysgalactiae in bovine mastitis and choose the appropriate antibiotics to treat mastitis.

Prevalence of Pathogenic Vibrio anguillarumAmong Oreochromis niloticusFish Fingerlings Infected with Saprolegniasis Around Qarun Lake

Vibrio anguillarum causes severe economic losses in fish and shellfish species and poses public health concerns. This study aimed to provide some information about the prevalence and virulence determinants of V. anguillarum isolated from the farmed Oreochromisniloticus fish fingerlings nearby Qarun Lake, El-Fayoum Governorate, Egypt. PCRtargeting the rpoA (RNA polymerase alpha subunit)gene was done forconfirmation of Vibriospp. Moreover, V. anguillarum isolates were biochemically identified and confirmed by 16S rRNA gene sequence.V. anguillarum isolates produced caseinase, gelatinase, lipase, lecithinase, haemolysins, and possessed biofilm formation. The virulence-related genes;empA, vah1, vah3 and vah4, flaA,angM, and angRwere detected in all tested isolates. Therefore, proper identification of V.anguillarumis crucial to better understand the ecology and distribution patterns of such pathogen within Egyptian aquaculture. Moreover, the water quality in these farms near Qarun Lake was less supportive for larval rearing activities of O. niloticus fish.

Genome Sequencing and Functional Characterization of Xanthomonas cucurbitae, the Causal Agent of Bacterial Spot Disease of Cucurbits

Bacterial leaf spot disease caused by Xanthomonas cucurbitae has severely affected the pumpkin industries in the Midwestern region of United States, with the bacteria mainly infecting pumpkin leaves and fruits, and leading to significant yield losses. In this study, we utilized genomics and genetics approaches to elucidate X. cucurbitae molecular mechanisms of pathogenesis during interaction with its host. We generated the first reference-quality whole-genome sequence of the X. cucurbitae type isolate and compared with other Xanthomonas species, X. cucurbitae has a smaller genome size with fewer virulence-related genes. RNA-seq analysis of X. cucurbitae under plant-mimicking media conditions showed altered transcriptional responses, with upregulation of virulence genes and downregulation of cellular homeostasis genes. Additionally, characterization of key virulence genes using gene deletion methods revealed that both type II enzymes and type III effectors are necessary for X. cucurbitae to cause infection in the pumpkin host.