Virulence Phenotypes Research Articles

Comparative genomic analysis reveals the potential transmission of Vibrio parahaemolyticus from freshwater food to humans

Vibrio parahaemolyticus is an increasingly important foodborne pathogen that cause acute gastroenteritis in humans. However, the prevalence and transmission of this pathogen in freshwater food remains unclear. This study aimed to determine the molecular characteristics and genetic relatedness of V. parahaemolyticus isolates obtained from freshwater food, seafood, environmental, and clinical samples. A total of 138 (46.6%) isolates were detected from 296 food and environmental samples, and 68 clinical isolates from patients. Notably, V. parahaemolyticus was more prevalent in freshwater food (56.7%, 85/150) than in seafood (38.8%, 49/137). Virulence phenotype analyses revealed that the high motility of isolates from freshwater food (40.0%) and clinical isolates (42.0%) was higher than that of isolates from seafood (12.2%), whereas the biofilm-forming capacity of freshwater food isolates (9.4%) was lower than that of seafood (22.4%) and clinical isolates (15.9%). Virulence genes analysis showed that 46.4% of the clinical isolates contained the tdh gene encoding thermostable direct hemolysin (TDH) and only two freshwater food isolates contained the trh gene encoding TDH-related hemolysin (TRH). Multilocus sequence typing (MLST) analysis divided the 206 isolates into 105 sequence types (STs), including 56 (53.3%) novel STs. ST2583, ST469, and ST453 have been isolated from freshwater food and clinical samples. Whole-genome sequence (WGS) analyses revealed that the 206 isolates were divided into five clusters. Cluster II contained isolates from freshwater food and clinical samples, whereas the other clusters contained isolates from seafood, freshwater food, and clinical samples. In addition, we observed that ST2516 had the same virulence pattern, with a close phylogenetic relationship to ST3. The increased prevalence and adaption of V. parahaemolyticus in freshwater food is a potential cause of clinical cases closely related to the consumption of V. parahaemolyticus contaminated freshwater food.

Virulence of Mycobacterium intracellulare clinical strains in a mouse model of lung infection – role of neutrophilic inflammation in disease severity

BackgroundMycobacterium intracellulare is a major etiological agent of Mycobacterium avium-intracellulare pulmonary disease (MAC-PD). However, the characteristics of the virulence of M. intracellulare and the in vivo chemotherapeutic efficacy remain unclear. In this study, we examined the virulence of nine M. intracellulare strains with different clinical phenotypes and genotypes in C57BL/6 mice.ResultsWe classified three types of virulence phenotypes (high, intermediate, and low) based on the kinetics of the bacterial load, histological lung inflammation, and neutrophilic infiltration. High virulence strains showed more severe neutrophilic infiltration in the lungs than intermediate and low virulence strains, with 6.27-fold and 11.0-fold differences of the average percentage of neutrophils in bronchoalveolar lavage fluid, respectively. In particular, the high virulence strain M.i.198 showed the highest mortality in mice, which corresponded to the rapid progression of clinical disease. In mice infected with the drug-sensitive high virulence strain M019, clarithromycin-containing chemotherapy showed the highest efficacy. Monotherapy with rifampicin exacerbated lung inflammation with increased lymphocytic and neutrophilic infiltration into the lungs.ConclusionsThe virulence phenotypes of clinical strains of M. intracellulare were diverse, with high virulence strains being associated with neutrophilic infiltration and disease progression in infected mice. These high virulence strains were proposed as a useful subject for in vivo chemotherapeutic experiments.

Antimicrobial resistance and genomic characterization of Salmonella enterica isolates from chicken meat.

This study investigated genotypic and phenotypic antimicrobial resistance profiles, phylogenic relatedness, plasmid and virulence composition of 39 Salmonella enterica strains isolated from chicken meat samples using whole genome sequencing (WGS) technology. Four distinct serotypes were identified; Salmonella Minnesota (16/39, 41%), Salmonella Infantis (13/39, 33.3%), Salmonella Enteritidis (9/39, 23.1%), and one isolate was detected for Salmonella Kentucky (1/39, 2.6%), with sequence types (STs) as followed: ST548, ST32, ST11, and ST198, respectively. Phenotypic resistance to tetracycline (91.2%), ampicillin (82.4%), sulfisoxazole (64.7%), and nalidixic acid (61.6%) was the most observed. Resistome analysis revealed the presence of resistance genes to aminoglycosides, β-lactamase, sulfonamides, trimethoprim, phenicol, lincosamide, macrolides, and tetracyclines. Plasmidome showed the presence of eight incompatibility groups, including IncA/C2, IncFIB(K)_1_Kpn3, Col440I_1, IncR, IncX1, IncI1_1_Alpha, IncFIB(S)/IncFII(S), IncHI2/IncHI2A, IncX2 and ColpVC plasmids across the 39 genomes. Three resistance genes, sul2, tetA and blaCMY-2, were predicted to be located on IncA/C2 plasmid in S. Minnesota isolates, whereas all S. Infantis isolates were positive to IncFIB(K)_1_Kpn3 plasmid that carries bla CTX-M-65 gene. Eleven Salmonella pathogenicity islands and up to 131 stress and/or virulence genes were identified in the evaluated genomes. Phylogenetic analysis showed four phylogroups that were consistent with the identified ST profiles with a high level of inter-diversity between isolates. This is the first genomic characterization of Salmonella isolates from retail chicken meat in Saudi Arabia using WGS technology. The availability of Salmonella genomes from multiple geographic locations, including Saudi Arabia, would be highly beneficial in future source-tracking, especially during epidemiological surveillance and outbreak investigations.

Genetic Variation of Puccinia triticina Populations in Iran from 2010 to 2017 as Revealed by SSR and ISSR Markers.

Puccinia triticina is a major wheat pathogen worldwide. Although Iran is within the Fertile Crescent, which is supposed to be the center of origin of both wheat and P. triticina, the knowledge of the genetic variability of local populations of this basidiomycete is limited. We analyzed 12 inter simple sequence repeats (ISSRs) and 18 simple sequence repeats (SSRs) of 175 P. triticina isolates sampled between 2010 and 2017 from wheat and other Poaceae in 14 provinces of Iran. SSRs revealed more polymorphisms than ISSRs, indicating they were more effective in differentiating P. triticina populations. Based on a dissimilarity matrix with a variable mutation rate for SSRs and a Dice coefficient for ISSRs, the isolates were separated into three large groups, each including isolates from diverse geographic origins and hosts. The grouping of SSR genotypes in UPGMA dendrograms was consistent with the grouping inferred from the Bayesian approach. However, isolates with a common origin clustered into separate subgroups within each group. The high proportion of heterozygous alleles suggests that in Iran clonal reproduction prevails over sexual reproduction of the pathogen. A significant correlation was found between SSR and ISSR genotypes and the virulence phenotypes of the isolates, as determined in a previous study.

Effects of Spatial Expression of Activating Transcription Factor 4 on the Pathogenicity of Two Phenotypes of Bovine Viral Diarrhea Virus by Regulating the Endoplasmic Reticulum-Mediated Autophagy Process.

The endoplasmic reticulum (ER) stress response is a highly conserved stress-defense mechanism and activates the adaptive unfolded protein response (UPR) to mitigate imbalance. The ER stress-activated signaling pathways can also trigger autophagy to facilitate cellular repair. Bovine viral diarrhea virus (BVDV) utilizes the host cellular ER as the primary site of the life cycle. However, the interplay between cellular ER stress and BVDV replication remains unclear. This report reveals that cytopathic (cp) and noncytopathic (ncp) BVDV have distinct strategies to regulate UPR mechanisms and ER stress-mediated autophagy for their own benefit. Immunoblot analysis revealed that cp and ncp BVDV differentially regulated the abundance of ER chaperone GRP78 for viral replication, while the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α)-activating transcription factor 4 (ATF4) pathway of the UPR was switched on at different stages of infection. Pretreatment with ER stress inducer promoted virion replication, but RNA interference (RNAi) knockdown of ATF4 in BVDV-infected cells significantly attenuated BVDV infectivity titers. More importantly, the effector ATF4 activated by cp BVDV infection translocated into the nucleus to mediate autophagy, but ATF4 was retained in the cytoplasm during ncp BVDV infection. In addition, we found that cp BVDV core protein was localized in the ER to induce ER stress-mediated autophagy. Overall, the potential therapeutic target ATF4 may contribute to the global eradication campaign of BVDV. IMPORTANCE The ER-tropic viruses hijack the host cellular ER as the replication platform of the life cycle, which can lead to strong ER stress. The UPR and related transcriptional cascades triggered by ER stress play a crucial role in viral replication and pathogenesis, but little is known about these underlying mechanisms. Here, we report that cytopathic and noncytopathic BVDV use different strategies to reprogram the cellular UPR and ER stress-mediated autophagy for their own advantage. The cytopathic BVDV unconventionally downregulated the expression level of GRP78, creating perfect conditions for self-replication via the UPR, and the noncytopathic BVDV retained ATF4 in the cytoplasm to provide an advantage for its persistent infection. Our findings provide new insights into exploring how BVDV and other ER-tropic viruses reprogram the UPR signaling pathway in the host cells for replication and reveal the attractive host target ATF4 for new antiviral agents.

Adaptation of Nontypeable Haemophilus influenzae in Human Airways in COPD: Genome Rearrangements and Modulation of Expression of HMW1 and HMW2.

Chronic obstructive pulmonary disease (COPD) is a common debilitating disorder that is the third most common cause of death globally. Chronic lower airway infection by nontypeable Haemophilus influenzae (NTHi) in adults with COPD increases airway inflammation, causes increased symptoms, and accelerates progressive loss of lung function. Little is known about the mechanisms by which NTHi survives in COPD airways. To explore this question, the present study analyzes, in detail, 14 prospectively collected, serial isolates of a strain that persisted for 543 days in a patient with COPD, including analysis of four gap-free complete genomes. The NTHi genome underwent inversion of a ~400-kb segment three times during persistence. This inversion event resulted in switching of expression of the HMW1A and HMW2A adhesins as the inversion sites are in the promoter regions of HMW1 and HMW2. Regulation of the level of expression of HMW 1 and HMW2 in the human airways was controlled by the ~400-kb inversion and by 7-bp repeats in the HMW promoters. Analysis of knockout mutants of the persistent strain demonstrated that HMW1 and HMW2 proteins both function in the adherence of NTHi to human respiratory epithelial cells during persistence and that HMW1 also facilitates invasion of epithelial cells. An inverse relationship between biofilm formation and HMW1 expression was observed during persistence. This work advances understanding of the mechanisms of persistence of NTHi in COPD airways, which can inform the development of novel interventions to treat and prevent chronic NTHi infection in COPD. IMPORTANCE Nontypeable Haemophilus influenzae (NTHi) persists in the lower airways of adults with chronic obstructive pulmonary disease (COPD) for months to years, increasing airway inflammation that accelerates the progressive loss of lung function. Understanding the mechanisms of persistence in human airways by NTHi is critical in developing novel interventions. Here, in detail, we studied longitudinally collected sequential isolates of a strain of NTHi that persisted in an adult with COPD, including analysis of four gap-free genomes and knockout mutants to elucidate how the genome adapts in human airways. The NTHi genome underwent a genome rearrangement during persistence and this inversion impacted regulation of expression of key virulence phenotypes, including adherence to respiratory epithelial cells, invasion of epithelial cells and biofilm formation. These novel observations advance our understanding of the mechanisms of persistence of NTHi in the airways of adults with COPD.

Phenotypic and Genotypic Investigation of Carbapenem-Resistant Acinetobacter baumannii in Maharaj Nakhon Si Thammarat Hospital, Thailand.

(1) Background: Acinetobacter baumannii is well known as a causative agent of severe hospital-acquired infections, especially in intensive care units. The present study characterised the genetic traits of biofilm-forming carbapenem-resistant A. baumannii (CRAB) clinical isolates. Additionally, this study determined the prevalence of biofilm-producing A. baumannii isolates from a tertiary care hospital and investigated the association of biofilms with the distribution of biofilm-related and antibiotic resistance-associated genotypes. (2) Methods: The 995 non-duplicate A. baumannii isolates were identified, and their susceptibilities to different antibiotics were determined using the disk diffusion method. Using the modified microtiter plate assay, the CRAB isolates were investigated for their biofilm formation ability. Hemolysin and protease activities were determined. CRABs were subjected to polymerase chain reaction (PCR) assays targeting blaVIM, blaNDM, blaIMP, blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, csuE and pgaB genes. Individual CRAB isolates were identified for their DNA fingerprint by repetitive element sequence-based (REP)-PCR. (3) Results: Among all A. baumannii isolates, 172 CRABs were identified. The major antibiotic resistance gene among the CRAB isolates was blaOXA-51-like (100%). Ninety-nine isolates (57.56%) were biofilm producers. The most prevalent biofilm gene was pgaB (79.65%), followed by csuE (76.74%). Evidence of virulence phenotypes revealed that all CRAB exhibited proteolytic activity; however, only four isolates (2.33%) were positive for the hemolytic-producing phenotype. REP-PCR showed that 172 CRAB isolates can be divided into 36-DNA fingerprint patterns. (4) Conclusions: The predominance of biofilm-producing CRAB isolates identified in this study is concerning. The characterisation of risk factors could aid in controlling the continual selection and spreading of the A. baumannii phenotype in hospitals, thereby improving patient care quality.

CRISPRi in Xanthomonas demonstrates functional convergence of transcription activator-like effectors in two divergent pathogens.

Functional analysis of large gene families in plant pathogens can be cumbersome using classical insertional mutagenesis. Additionally, Cas9 toxicity has limited the application of CRISPR-Cas9 for directed mutagenesis in bacteria. Here, we successfully applied a CRISPR interference strategy to investigate the cryptic role of the transcription activator-like effector (tale) multigene family in several plant-pathogenic Xanthomonas bacterial species, owing to their contribution to pathogen virulence. Single guide RNAs (sgRNAs) designed against Xanthomonas phaseoli pv manihotis tale conserved gene sequences efficiently silenced expression of all tales, with concomitant decrease in virulence and TALE-induced host gene expression. The system is readily translatable to other Xanthomonas species infecting rice, citrus, Brassica, and cassava, silencing up to 16 tales in a given strain using a single sgRNA. Complementation with plasmid-borne designer tales lacking the sgRNA-targeted sequence restored molecular and virulence phenotypes in all pathosystems. Our results evidenced that X. campestris pv campestris CN08 tales are relevant for symptom development in cauliflower. They also show that the MeSWEET10a sugar transporter is surprisingly targeted by the nonvascular cassava pathogen X. cassavae, highlighting a new example of TALE functional convergence between phylogenetically distant Xanthomonas. Overall, this novel technology provides a platform for discovery and rapid functional understanding of highly conserved gene families.

Regulation of Virus Replication by BK Polyomavirus Small T Antigen.

Polyomavirus small T antigen (tAg) plays important roles in regulating viral replication, the innate immune response, apoptosis, and transformation for SV40, Merkel cell polyomavirus (MCPyV), murine polyomavirus (MuPyV), and JC polyomavirus (JCPyV). However, the function of BK polyomavirus (BKPyV) tAg has been much less studied. Here, we constructed mutant viruses that do not express tAg, and we showed that, in contrast with other polyomaviruses, BKPyV tAg inhibits large T antigen (TAg) gene expression and viral DNA replication. However, this occurs only in an archetype viral background. We also observed that the transduction of cells with a lentivirus-expressing BKPyV tAg kills the cells. We further discovered that BKPyV tAg interacts not only with PP2A A and C subunits, as has been demonstrated for other polyomavirus tAg proteins, but also with PP2A B''' subunit members. Knocking down either of two B''' subunits, namely STRN or STRN3, mimics the phenotype of the tAg mutant virus. However, a virus containing a point mutation in the PP2A binding domain of tAg only partially affected virus TAg expression and DNA replication. These results indicate that BKPyV tAg downregulates viral gene expression and DNA replication and that this occurs in part through interactions with PP2A. IMPORTANCE BK polyomavirus is a virus that establishes a lifelong infection of the majority of people. The infection usually does not cause any clinical symptoms, but, in transplant recipients whose immune systems have been suppressed, unchecked virus replication can cause severe disease. In this study, we show that a viral protein called small T antigen is one of the ways that the virus can persist without high levels of replication. Understanding which factors control viral replication enhances our knowledge of the virus life cycle and could lead to potential interventions for these patients.

Genome Analysis of Two Pseudomonas syringae pv. aptata Strains with Different Virulence Capacity Isolated from Sugar Beet: Features of Successful Pathogenicity in the Phyllosphere Microbiome.

Members of the Pseudomonas syringae species complex are heterogeneous bacteria that are the most abundant bacterial plant pathogens in the plant phyllosphere, with strong abilities to exist on and infect different plant hosts and survive in/outside agroecosystems. In this study, the draft genome sequences of two pathogenic P. syringae pv. aptata strains with different in planta virulence capacities isolated from the phyllosphere of infected sugar beet were analyzed to evaluate putative features of survival strategies and to determine the pathogenic potential of the strains. The draft genomes of P. syringae pv. aptata strains P16 and P21 are 5,974,057 bp and 6,353,752 bp in size, have GC contents of 59.03% and 58.77%, respectively, and contain 3,439 and 3,536 protein-coding sequences, respectively. For both average nucleotide identity and pangenome analysis, P16 and P21 largely clustered with other pv. aptata strains from the same isolation source. We found differences in the repertoire of effectors of the type III secretion system among all 102 selected strains, suggesting that the type III secretion system is a critical factor in the different virulent phenotypes of P. syringae pv. aptata. During genome analysis of the highly virulent strain P21, we discovered genes for T3SS effectors (AvrRpm1, HopAW1, and HopAU1) that were not previously found in genomes of P. syringae pv. aptata. We also identified coding sequences for pantothenate kinase, VapC endonuclease, phospholipase, and pectate lyase in both genomes, which may represent novel effectors of the type III secretion system. IMPORTANCE Genome analysis has an enormous effect on understanding the life strategies of plant pathogens. Comparing similarities with pathogens involved in other epidemics could elucidate the pathogen life cycle when a new outbreak happens. This study represents the first in-depth genome analysis of Pseudomonas syringae pv. aptata, the causative agent of leaf spot disease of sugar beet. Despite the increasing number of disease reports in recent years worldwide, there is still a lack of information about the genomic features, epidemiology, and pathogenic life strategies of this particular pathogen. Our findings provide advances in disease etiology (especially T3SS effector repertoire) and elucidate the role of environmental adaptations required for prevalence in the pathobiome of the sugar beet. From the perspective of the very heterogeneous P. syringae species complex, this type of analysis has specific importance in reporting the characteristics of individual strains.

Blalock-Taussig Shunt Blockage Caused by Small Colony Variants of Enterococcus faecalis.

To the Editors: Small colony variants (SCVs) constitute a subpopulation of nutrient-demanding bacteria that grow slower than the wild type. Selection pressure exerted by environmental factors, such as antimicrobial agents.1,2 SCVs of Enterococcus sp. are less frequent. We report the first case of Blalock–Taussig shunt (BTS) blockage caused by SCVs of Enterococcus faecalis. A 1-year-old boy diagnosed with tetralogy of Fallot underwent left BTS surgery and closure of patent ductus arteriosus 10 months before presentation. On postoperative day 11, a catheter-related bloodstream infection (CRBSI) was caused by E. faecalis. The peripherally inserted central catheter was removed. Echocardiography revealed the absence of verrucae, and the modified Duke criteria were not definite. Ampicillin was administered for 2 weeks; 4 months postcompletion of this therapy, the left BTS was occluded, and a right BTS surgery was performed with the left BTS remaining in place. Although he underwent this surgery for endocardial repair, the left BTS was only removed during the operation, as pus was found in the occluded left BTS during the intraoperative manipulation. No bacteria were detected in the pus using Gram staining, but vancomycin was administered for suspected shunt infection. After 2 days of culture, colonies of approximately 0.2–1 mm and 2–3 mm diameters were observed on the blood agar medium maintained in an aerobic environment and the Brucella HK agar (Kyokuto, Inc. Tokyo, Japan) medium maintained in an anaerobic environment, respectively (Fig. 1). Although biochemical analysis indicated Enterococcus spp., identification and drug sensitivity test performed using Micro Scan Walkaway 40 plus (Beckman Coulter, Inc. CA) indicated poor growth after 24 hours of incubation. Matrix-assisted laser desorption/ionization–time-of-flight analysis indicated the presence of E. faecalis; hence, the antimicrobial was changed to ampicillin, which was administered for 4 weeks to control the mediastinitis. He was discharged without sequelae and had no recurrence of bacteremia for over a year.FIGURE 1.: Bacterial colonies cultured using blood agar and Brucella HK agar media.SCVs originated due to stresses, such as perioperative exposure to antimicrobials used to treat E. faecalis bacteremia. In addition, the short duration of antimicrobial therapy used to treat CRBSI may be another reason. Enterococcus spp. bacteremia is considerably associated with the occurrence of infectious endocarditis (IE) caused by a prosthetic valve; hence, echocardiography is recommended.3 In this case, antimicrobials were administered for 2 weeks. However, 4–6 weeks of antimicrobial therapy may be appropriate for treating IE. SCVs, with a typical phenotype of low virulence and long-term persistence, adapt to the intracellular environment more efficiently than the wild type. We have listed previously reported cases of SCVs of Enterococcus spp. (Table Supplemental Digital Content 1, https://links.lww.com/INF/E930,); half of which were IE, including two cases of IE related to prosthetic valves, which may be attributed to the fact that SCVs tend to form biofilms, leading to infection in prostheses.4 Moreover, Enterococcus spp. have higher probability of forming biofilms than other bacteria. E. faecalis is particularly more prone to biofilm formation than E. faecium.5 This case indicates that SCVs of E. faecalis may survive for a long period, especially in case of artifact infection.

Phylogenetic Analysis and Virulence Characteristics of Methicillin-Resistant Staphylococcus aureus ST45 in China: a Hyper-Virulent Clone Associated with Bloodstream Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 45 (ST45) was rarely found in China. This study was conducted to trace the transmission and evolution of emerging MRSA ST45 strains in mainland China and explore its virulence. A total of 27 ST45 isolates were included for whole-genome sequencing and genetic characteristic analysis. Epidemiological results showed that MRSA ST45 isolates were often obtained from blood, primarily originated in Guangzhou, and carried diverse virulence and drug resistance genes. Staphylococcal cassette chromosome mec type IV (SCCmec IV) dominated in MRSA ST45 (23/27, 85.2%). ST45-SCCmec V was located on a phylogenetic clade distinct from the SCCmec IV cluster. We selected two representative isolates, MR370 (ST45-SCCmec IV) and MR387 (ST45-SCCmec V), and performed hemolysin activity, a blood killing assay, a Galleria mellonella infection model, and a mouse bacteremia model, as well as real-time fluorescence quantitative PCR. MR370 was proved to have extreme virulence in the phenotypic assays and at the mRNA level compared with ST59, ST5, and USA300 MRSA strains. MR387 was comparable to USA300-LAC on the phenotype and was verified to have higher expression of scn, chp, sak, saeR, agrA, and RNAIII than USA300-LAC. The results emphasized the extraordinary performance of MR370 and the good potential of MR387 in virulence causing bloodstream infection. Meanwhile, we conclude that China MRSA ST45 showed two different clonotypes, which may be widespread in the future. The entire study is valuable as a timely reminder and reports virulence phenotypes of China MRSA ST45 for the first time. IMPORTANCE Methicillin-resistant Staphylococcus aureus ST45 is epidemic worldwide. This study contributed to the awareness of the Chinese hyper-virulent MRSA ST45 strains and served as a timely reminder of its wide dissemination of clonotypes. Further, we provide novel insights for prevention from the perspective of bloodstream infections. ST45-SCCmec V is a clonotype deserving special attention in China, and we performed genetic and phenotypic analyses for the first time on it.

StkP- and PhpP-Mediated Posttranslational Modifications Modulate the S. pneumoniae Metabolism, Polysaccharide Capsule, and Virulence.

Pneumococcal Ser/Thr kinase (StkP) and its cognate phosphatase (PhpP) play a crucial role in bacterial cytokinesis. However, their individual and reciprocal metabolic and virulence regulation-related functions have yet to be adequately investigated in encapsulated pneumococci. Here, we demonstrate that the encapsulated pneumococcal strain D39-derived D39ΔPhpP and D39ΔStkP mutants displayed differential cell division defects and growth patterns when grown in chemically defined media supplemented with glucose or nonglucose sugars as the sole carbon source. Microscopic and biochemical analyses supported by RNA-seq-based global transcriptomic analyses of these mutants revealed significantly down- and upregulated polysaccharide capsule formation and cps2 genes in D39ΔPhpP and D39ΔStkP mutants, respectively. While StkP and PhpP individually regulated several unique genes, they also participated in sharing the regulation of the same set of differentially regulated genes. Cps2 genes were reciprocally regulated in part by the StkP/PhpP-mediated reversible phosphorylation but independent of the MapZ-regulated cell division process. StkP-mediated dose-dependent phosphorylation of CcpA proportionately inhibited CcpA-binding to Pcps2A, supporting increased cps2 gene expression and capsule formation in D39ΔStkP. While the attenuation of the D39ΔPhpP mutant in two mouse infection models corroborated with several downregulated capsules-, virulence-, and phosphotransferase systems (PTS)-related genes, the D39ΔStkP mutant with increased amounts of polysaccharide capsules displayed significantly decreased virulence in mice compared to the D39 wild-type, but more virulence compared to D39ΔPhpP. NanoString technology-based inflammation-related gene expression and Meso Scale Discovery-based multiplex chemokine analysis of human lung cells cocultured with these mutants confirmed their distinct virulence phenotypes. StkP and PhpP may, therefore, serve as critical therapeutic targets.

Cheminformatics Bioprospection of Sunflower Seeds' Oils against Quorum Sensing System of Pseudomonas aeruginosa.

Clinically significant pathogens such as Pseudomonas aeruginosa evade the effects of antibiotics using quorum sensing (QS) systems, making antimicrobial resistance (AMR) a persistent and potentially fatal global health issue. Hence, QS has been identified as a novel therapeutic target for identifying novel drug candidates against P. aeruginosa, and plant-derived products, including essential oils, have been demonstrated as effective QS modulators. This study assessed the antipathogenic efficacy of essential oils from two sunflower cultivars (AGSUN 5102 CLP and AGSUN 5106 CLP) against P. aeruginosa ATCC 27853 in vitro and in silico. At the sub-inhibitory concentrations, both AGSUN 5102 CLP (62.61%) and AGSUN 5106 CLP (59.23%) competed favorably with cinnamaldehyde (60.74%) and azithromycin (65.15%) in suppressing the expression of QS-controlled virulence phenotypes and biofilm formation in P. aeruginosa. A further probe into the mechanism of anti-QS action of the oils over a 100-ns simulation period against Las QS system revealed that phylloquinone (-66.42 ± 4.63 kcal/mol), linoleic acid (-53.14 ± 3.53 kcal/mol), and oleic acid (-52.02 ± 3.91 kcal/mol) had the best affinity and structural compactness as potential modulators of LasR compared to cinnamaldehyde (-16.95 ± 1.75 kcal/mol) and azithromycin (-32.08 ± 10.54 kcal/mol). These results suggest that the identified compounds, especially phylloquinone, could be a possible LasR modulator and may represent a novel therapeutic alternative against infections caused by P. aeruginosa. As a result, phylloquinone could be further studied as a QS modulator and perhaps find utility in developing new therapeutics.

Genetic characteristics and linkage of virulence genes of the Puccinia striiformis f. sp. tritici TSA-6 isolate to Yr5 host resistance.

To understand inheritance of the TSA-6 Puccinia striiformis f. sp. tritici isolate, virulent to Yr5, that was recently detected in China, we analyzed avirulence and virulence of 120 selfed progeny lines from Berberis shensiana. The results showed that the TSA-6 isolate is virulent against the Yr5 resistance gene, and overall progeny lines were categorized into 73 virulence phenotypes (VPs), and of these, 72 VPs differed from the isolate TSA-6, and only 1 VP including 3 progeny was identical to the parental isolate. The analyses indicated that the TSA-6 isolate is homozygous for avirulence at Yr10, Yr15 and Yr26 resistance loci, and virulence at YrA resistance locus. The TSA-6 isolate is heterozygous for avirulence at the Yr2, Yr3, Yr5, Yr7 and Yr8 resistance loci, which are controlled by a dominant/recessive relationship. The Yr1, Yr6, Yr9, Yr17, Yr27, Yr25, Yr28, Yr29, Yr32, YrTr1 and YrSP resistance loci are governed by two complementary dominant/recessive genes. Avirulence against heterozygous Yr4, Yr43，Yr44, Yr76 and YrExp2 resistance loci is regulated by a dominant and recessive, or a dominant and suppressor gene pairs. Totally, 117 multi-locus genotypes (MLGs) were detected at 24 KASP-SNP marker loci among the 120 progeny. Using these marker loci, we constructed a linkage map with a genetic distance interval spanning 624.5 cM. Quantitative trait loci (QTL) corresponding to phenotypic segregation for virulence at 20 Yr resistance loci in addition to the Yr1 resistance locus were identified. These results facilitate our understanding of Pst virulence evolution, and simplify breeding of wheat cultivars with effective resistance to wheat stripe rust.

Virulence Phenotyping and Molecular Genotyping Reveal High Diversity Within and Strong Gene Flow Between the Puccinia striiformis f. sp. tritici Populations Collected from Barberry and Wheat in Shaanxi Province of China.

Emergence of new Puccinia striiformis f. sp. tritici races that overcome resistance of wheat cultivars is a challenging issue for wheat production. Although sexual reproduction of the fungus on barberry plants under field conditions in the spring in China has been reported, the diversity of the pathogen on barberry plants and the relationship to the population in wheat fields have not been determined. In the present study, two P. striiformis f. sp. tritici populations collected in western Shaanxi Province in May 2016, one from barberry plants (103 isolates) and the other from nearby wheat crops (107 isolates), were phenotyped for virulence and genotyped with simple sequence repeat (SSR) markers. The phenotypic and genotypic data of the two populations were compared to determine their relationships. A total of 120 races, including 29 previously known races (seven were shared by the two populations) and 91 new races (35 from barberry and 56 from wheat), were identified. Similarly, a total of 132 multilocus genotypes, including 51 only from barberry, 77 only from wheat, and four from both, were detected using the SSR markers. Analyses of molecular variance identified high (93%) genetic variance within populations and low but still significant variance (7%) between the populations. Nonparametric multivariate discriminant analysis of principal components and STRUCTURE analysis showed that the two populations had a close relationship with little genetic differentiation (FST = 0.038) and strong gene flow (Nm = 6.34, P = 0.001) between them. Although the analysis of linkage disequilibrium indicated clonal populations, the isolation of P. striiformis f. sp. tritici from barberry plants and the high genetic diversities in the barberry and wheat populations suggest that barberry plants provide aeciospores to infect wheat crops in the area. The information is useful for understanding stripe rust epidemiology and management of the disease.

Interactions between insect vectors and plant pathogens span the parasitism-mutualism continuum.

Agricultural crops infected with vector-borne pathogens can suffer severe negative consequences, but the extent to which phytopathogens affect the fitness of their vector hosts remains unclear. Evolutionary theory predicts that selection on vector-borne pathogens will favour low virulence or mutualistic phenotypes in the vector, traits facilitating effective transmission between plant hosts. Here, we use a multivariate meta-analytic approach on 115 effect sizes across 34 unique plant-vector-pathogen systems to quantify the overall effect of phytopathogens on vector host fitness. In support of theoretical models, we report that phytopathogens overall have a neutral fitness effect on vector hosts. However, the range of fitness outcomes is diverse and span the parasitism-mutualism continuum. We found no evidence that various transmission strategies, or direct effects and indirect (plant-mediated) effects, of phytopathogens have divergent fitness outcomes for the vector. Our finding emphasizes diversity in tripartite interactions and the necessity for pathosystem-specific approaches to vector control.

Characterizing a century of genetic diversity and contemporary antigenic diversity of N1 neuraminidase in influenza A virus from North American swine.

Influenza A viruses (IAVs) of the H1N1 classical swine lineage became endemic in North American swine following the 1918 pandemic. Additional human-to-swine transmission events after 1918, and a spillover of H1 viruses from wild birds in Europe, potentiated a rapid increase in genomic diversity via reassortment between introductions and the endemic classical swine lineage. To determine mechanisms affecting reassortment and evolution, we conducted a phylogenetic analysis of N1 and paired HA swine IAV genes in North America between 1930 and 2020. We described fourteen N1 clades within the N1 Eurasian avian lineage (including the N1 pandemic clade), the N1 classical swine lineage, and the N1 human seasonal lineage. Seven N1 genetic clades had evidence for contemporary circulation. To assess antigenic drift associated with N1 genetic diversity, we generated a panel of representative swine N1 antisera and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assays and antigenic cartography. Within the N1 genes, the antigenic similarity was variable and reflected shared evolutionary history. Sustained circulation and evolution of N1 genes in swine had resulted in a significant antigenic distance between the N1 pandemic clade and the classical swine lineage. Between 2010 and 2020, N1 clades and N1-HA pairings fluctuated in detection frequency across North America, with hotspots of diversity generally appearing and disappearing within 2 years. We also identified frequent N1-HA reassortment events (n = 36), which were rarely sustained (n = 6) and sometimes also concomitant with the emergence of new N1 genetic clades (n = 3). These data form a baseline from which we can identify N1 clades that expand in range or genetic diversity that may impact viral phenotypes or vaccine immunity and subsequently the health of North American swine.

QCR7 affects the virulence of Candida albicans and the uptake of multiple carbon sources present in different host niches.

Candida albicans is a commensal yeast that may cause life-threatening infections. Studies have shown that the cytochrome b-c1 complex subunit 7 gene (QCR7) of C. albicans encodes a protein that forms a component of the mitochondrial electron transport chain complex III, making it an important target for studying the virulence of this yeast. However, to the best of our knowledge, the functions of QCR7 have not yet been characterized. A QCR7 knockout strain was constructed using SN152, and BALb/c mice were used as model animals to determine the role of QCR7 in the virulence of C. albicans. Subsequently, the effects of QCR7 on mitochondrial functions and use of carbon sources were investigated. Next, its mutant biofilm formation and hyphal growth maintenance were compared with those of the wild type. Furthermore, the transcriptome of the qcr7Δ/Δ mutant was compared with that of the WT strain to explore pathogenic mechanisms. Defective QCR7 reduced recruitment of inflammatory cells and attenuated the virulence of C. albicans infection in vivo. Furthermore, the mutant influenced the use of multiple alternative carbon sources that exist in several host niches (GlcNAc, lactic acid, and amino acid, etc.). Moreover, it led to mitochondrial dysfunction. Furthermore, the QCR7 knockout strain showed defects in biofilm formation or the maintenance of filamentous growth. The overexpression of cell-surface-associated genes (HWP1, YWP1, XOG1, and SAP6) can restore defective virulence phenotypes and the carbon-source utilization of qcr7Δ/Δ. This study provides new insights into the mitochondria-based metabolism of C. albicans, accounting for its virulence and the use of variable carbon sources that promote C. albicans to colonize host niches.

In Vivo Effect of a Nisin–Biogel on the Antimicrobial and Virulence Signatures of Canine Oral Enterococci

Periodontal disease is a relevant oral disease in dogs and nisin-biogel has been previously proposed to be used in its control. Enterococci, as inhabitants of the oral cavity with a high genetic versatility, are a reliable bacterial model for antimicrobial studies. Our goal was to evaluate the in vivo influence of the long-term dental application of the nisin-biogel on the virulence and antimicrobial signatures of canine oral enterococci. Twenty dogs were randomly allocated to one of two groups (treatment group-TG with nisin-biogel dental application, or control group-CG without treatment) and submitted to dental plaque sampling at day 0 and after 90 days (T90). Samples were processed for Enterococcus spp. isolation, quantification, identification, molecular typing and antimicrobial and virulence characterization. From a total of 140 enterococci, molecular typing allowed us to obtain 70 representative isolates, mostly identified as E. faecalis and E. faecium. No significant differences (p > 0.05) were observed in the virulence index of the isolates obtained from samples collected from the TG and CG at T90. At T90, a statistically significant difference (p = 0.0008) was observed in the antimicrobial resistance index between the isolates from the TC and CG. Oral enterococci were revealed to be reservoirs of high resistant and virulent phenotypes.