Virulent Group Research Articles

Multiplex PCR screening for virulence genes of Klebsiella pneumoniae isolated from microbiota of diseased and healthy people

Introduction. Klebsiella pneumoniae in human microbiota may appear as a part of commensal microbiota, and as hypervirulent pathogen, for example, hypermucoid pathotype. This pathotype is characterized by certain genetic determinants, leading to the possibility of detecting the pathogenic potential of isolates by PCR. Aim of the study: to evaluate and compare pathogenic potential of K. pneumoniae isolates from practically healthy people, patients with inflammatory bowel disease (IBD) and extraintestinal infections (ExII).Materials and methods. Testing was performed with the set of nucleotides for multiplex PCR analysis targeting eight potentially virulent genes with the following functions: ferrum uptake (ybsT, kfu, iutA), adhesion and invasion (mrkD), hypermucoid phenotype and virulent serotypes (mrkD, magA, rmpA, k2) and metabolism of allantoin (allS). PCR assay was used to screen Klebsiella pneumoniae isolates from feces of patients with IBD (69 isolates) and of practically healthy people (68 isolates), and multiresistant isolates from biological material (blood, urine, surgical wounds, bronchoalveolar lavage) of patients with extraintestinal infections (mrExII, 25 isolates).Results. Results of the testing demonstrated association of four of targeted determinants with the patients diagnoses. YbtS gene was significantly more often found in isolates from IBD (р = 0.024) and mrExII (p 0.001) groups. RmpA gene was significantly more often detected in IBD group (р = 0.038). Extraintestinal infectious isolates were significantly (р ≤ 0.001) enriched with mrkD and allS genes (р = 0.032).Conclusion. The most potentially virulent group was isolated from patients with extraintestinal infections, the least virulent — isolates from feces of practically healthy people. The most frequently detected virulence genes were involved in adhesion and hypermucoid phenotype formation.

Genotypic and phenotypic quantitative microbial risk assessment model of human salmonellosis related to the consumption of chicken meat in the central region of Mexico

Chicken meat is often associated withSalmonella entericacontamination worldwide. This study proposes a risk assessment model for human salmonellosis linked to the domestic consumption of chicken meat in the central region of Mexico, incorporating genotypic and phenotypic data. SixS. entericagroups were used, considering the presence of specific virulence genes and multidrug resistance (MDR). Sixteen exposure scenarios were established considering retail point (RP1 = fresh market/butcher shop; RP2 = mini-super/supermarket), transportation, home storage, cooking, and cross-contamination. The model predicted a mean annual salmonellosis cases of 66,754 due to chicken consumption (CI95% 10775–231606). The mean probability of illness (Pill) among the exposure scenarios ranged from 2.5 × 10-9 to 3.7 × 10-6, 7.7 × 10-8 to 1.1 × 10-4, and 6.7 × 10-4 to 7.8 × 10-2 for low, moderate, and high virulence groups. Exposure scenarios with the highest Pill were not responsible for most cases due to their low frequency of occurrence. The high virulence/ MDR group was responsible for most cases (66.5 %), despite the low S. enterica prevalence (RP1 0.5 % and RP2 5.0 %). The years lost due to disability (YLD) value for MDR was 2.6 × higher than for non-MDR. Spearman rank showed that the inputs with higher influence on the variability of salmonellosis depended on the type of exposure scenario. For example, the cooking temperature and time had the most significant influence in the scenarios where S. enterica can survive after cooking.Including the microbial genotypic and phenotypic characteristics in risk assessment modeling highlights the importance of focusing on high-virulent and MDR strains, which are not the most frequent but represent the highest public health risk.

Elucidating the virulence genes harboured by carbapenemase- and non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae rectal isolates from patients admitted to intensive care units using whole-genome sequencing in Kuwait.

Introduction. Klebsiella pneumoniae is a Gram-negative pathogen responsible for community- and nosocomial-acquired infections. The presence of an accessory genome determines the bacterial pathogenicity and the host immune response, and thus indicates multidrug-resistant strains or more virulent groups. Little is known about the virulence genes in K. pneumoniae in Kuwait.Hypothesis/Gap Statement. The diversity of virulence genes and capsule loci in K. pneumoniae isolates warrants further genomic studies to better understand their transmission within the hospitals in Kuwait.Aim. We aimed to investigate the virulence genes harboured by K. pneumoniae isolated from rectal swabs of intensive care unit (ICU) patients in two Kuwaiti teaching hospitals.Methodology. Six isolates from patients in the ICUs of Al Razi and Mubarak hospitals, designated RZH144, RZH132 RZH108 and RZH173, and MKH381 and MKH347, respectively, were subjected to whole-genome sequencing (WGS) assays. RZH144 and RZH132 were non-carbapenemase-producing K. pneumoniae (NCKP) isolates negative for genes encoding carbapenemase production by PCR assays, and the remaining four were carbapenemase-producing K. pneumoniae (CPKP) isolates. Isolates were characterized by phenotypic, PCR and WGS methods. Susceptibility testing was performed by E test and clonality by multilocus sequence typing. Analysis of the isolates' assembled contigs was carried out using Kleborate (https://pathogen.watch).Results. An NCPE RZH132 K. pneumoniae isolate belonged to ST231-wzi104 and harboured gene clusters encoding the biosynthesis of the siderophore aerobactin (iuc5) on 62-3LV. The capsular locus variants were KL51 and O locus O1v2. Another NCPKP RZH144 isolate was confirmed as ST43-wzi412 and harboured KL61 and O1v1. The four CPKP isolates harboured two virulence loci - ybt14 and iuc5 - encoding the siderophores yersiniabactin and aerobactin, respectively. They belonged to ST231-wzi104 and harboured yersiniabactin on ICEKp5. The sequence type of ybt was YbST145-1LV. Strain RZ108 was devoid of virulence loci. Its sequence type was ST15-wzi151 and harboured KL48 and O1V1. ST231 clonal lineage isolates shared common virulence plasmid variants.Conclusion. The CPKP ST231 had the highest virulence score and contained iuc5, which was found for the first time in ST231-CPKP isolates in Kuwait.

Use of Large-Scale Genomics to Identify the Role of Animals and Foods as Potential Sources of Extraintestinal Pathogenic Escherichia coli That Cause Human Illness

Extraintestinal pathogenic Escherichia coli (ExPEC) cause urinary tract and potentially life-threatening invasive infections. Unfortunately, the origins of ExPEC are not always clear. We used genomic data of E. coli isolates from five U.S. government organizations to evaluate potential sources of ExPEC infections. Virulence gene analysis of 38,032 isolates from human, food animal, retail meat, and companion animals classified the subset of 8142 non-diarrheagenic isolates into 40 virulence groups. Groups were identified as low, medium, and high relative risk of containing ExPEC strains, based on the proportion of isolates recovered from humans. Medium and high relative risk groups showed a greater representation of sequence types associated with human disease, including ST-131. Over 90% of food source isolates belonged to low relative risk groups, while >60% of companion animal isolates belonged to medium or high relative risk groups. Additionally, 18 of the 26 most prevalent antimicrobial resistance determinants were more common in high relative risk groups. The associations between antimicrobial resistance and virulence potentially limit treatment options for human ExPEC infections. This study demonstrates the power of large-scale genomics to assess potential sources of ExPEC strains and highlights the importance of a One Health approach to identify and manage these human pathogens.

Prevalence of extended-spectrum β-lactamase-producing Enterobacterales in retail sheep meat from Zagazig city, Egypt

BackgroundThe goal of this study was to investigate the prevalence of extended-spectrum β-lactamase production in Enterobacterales isolated from retail sheep meat in Zagazig, Egypt.MethodsOne hundred random samples of sheep meat were collected from different retail butcher shops (n = 5) in the city of Zagazig, Egypt. Bacterial isolates were identified by MALDI-TOF MS and screened for antibiotic susceptibility by disk diffusion; further genotypic characterization of β-lactamase-encoding genes was performed with Real-Time PCR. E. coli strains were phylotyped with the Clermont triplex PCR method.ResultsOf the total of 101 bacterial isolates recovered from retail sheep meat samples, 93 were E. coli, six were Enterobacter cloacae and two were Proteus mirabilis. As many as 17% of these 100 samples showed ESBL phenotypes, all were E. coli. The blaCTX-M genes were detected in seven isolates (six were blaCTX-M-15 and one was blaCTX-M-14), three isolates harboured blaTEM (all were blaTEM-one), and two carried genes of the blaSHV family (both were blaSHV-12). Eight E. coli isolates expressed ESBL phenotype but no blaTEM, blaSHV or blaCTX-M genes were detected by PCR. ESBL- positive E. coli isolates were nearly equally distributed over the commensal groups A/B1 and the virulent group D.ConclusionNearly one in five sheep meat samples was contaminated with ESBL-E. coli. This further corroborates the potential role played by contaminated meat in the increasing resistance rates that have been reported worldwide.

Development and validation of high-resolution melting assays for the detection of potentially virulent strains of Escherichia coli O103 and O121

Virulent strains of Shiga toxin-producing Escherichia coli (STEC) serogroups O103 and O121 are considered adulterants in beef. Two high-resolution melting (HRM) real-time PCR assays were standardized for the specific detection and discrimination of potentially virulent and avirulent strains of E. coli O103 and O121. The O103 HRM assay offered the possibility to distinguish clearly STEC O103:H2 from STEC O103:H25. The two standardized assays were extensively validated using 215 pure culture strains, laboratory inoculated food samples, and naturally contaminated beef (n = 84) and pork (n = 84) enrichments collected from the red meat surveillance program. Both HRM assays showed 100% inclusivity and exclusivity using pure culture strains and enriched spiked food samples. Data from this study shows the ability of the standardized assays to specifically detect the strains of each target serogroup and, most importantly, to differentiate the strains present into potentially virulent or avirulent groups. The assays standardized in this study can be helpful for food surveillance programs and help mitigate product loss due to the presence of avirulent strains lacking crucial virulence genes (stx and eae).

Virulence and phylogenetic groups of Escherichia coli cultured from raw sewage in Kuwait

In Kuwait, some untreated sewage is discharged into the sea which poses health risks. Therefore, we determined the virulence traits and the phylogenetic groups of E. coli cultured from raw sewage. Sewage was collected once every month for 12 months with culturing of a total of 140 E. coli isolates. E. coli was typed by the methods of Clermont. The five pathotypes of diarrheagenic E. coli (DEC), and extra-intestinal pathogenic E. coli (ExPEC) were detected by specific PCR assays. Four virulence genes which correlate with pathogenicity in animal models, were used for the first time for detection of ExPEC—vat (vacuolating auto-transporter toxin), fyuA (yersiniabactin receptor), chuA (heme-binding protein), and yfcV (major subunit of putative chaperon-usher fimbria). Most E. coli belonged to phylogenetic groups A (65[45%]) and B1 (34[24.3%]). Three (2.1%) isolates were DEC, while 14 (10%) isolates were ExPEC mostly in group B2 (57.1%). A relatively high prevalence of ExPEC in sewage has public health implications.

Genetic Diversity and Population Structure Reveal Cryptic Genetic Variation and Long Distance Migration of Puccinia graminis f. sp. tritici in the Indian Subcontinent.

Stem rust caused by Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of wheat worldwide since time immemorial. Several wheat stem rust outbreaks have been reported worldwide including India. Approximately 7 mha wheat area in central and peninsular India is highly vulnerable to stem rust epidemics. In this study, a repository of 29 single genotype uredospore pathotypes, representing five geographical regions, was characterized by investigating their virulence phenotype and simple sequence repeat (SSR) genotypes using 37 reproducible polymorphic SSR markers, 32 of which had ≥ 0.50 polymorphic information content (PIC) value. Virulence phenotypes were used to evaluate the virulence frequency (VF) and construct a hypothetical evolutionary hierarchy of these pathotypes. We projected seven lineages to explain the evolutionary pattern of the Pgt population. The VF of these pathotypes ranged between 0% and 100%. The virulence-based neighbor-joining (NJ) cluster analysis grouped Pgt pathotypes into five virulence groups. Likewise, five molecular groups were categorized using molecular genotypes. The molecular grouping was supported by principal coordinate analysis (PCoA), which revealed 25% of the cumulative variance contributed by the first two axes. Analysis of molecular variance (AMOVA) revealed 8 and 92% of the variation among and within the populations, respectively. The Mantel test confirmed a positive but weak correlation (R2 = 0.15) between virulence phenotypes and SSR genotypes. The highest and lowest values of different genetic diversity parameters (Na, Ne, I, He, uHe, and %P) revealed maximum and minimum variability in the Pgt population from Maharashtra and Uttar Pradesh, respectively. The population structure analysis clustered 29 Pgt pathotypes into two subpopulations and an admixture. Our results demonstrated that there was significant genetic diversity among Pgt pathotypes resulting from their long-distance dispersal ability complemented by gene flow. These findings provide insights into the virulence patterns, genetic variations, and possible evolution of Pgt pathotypes, which would support strategic stem rust resistance breeding.

Pathotype Identification and Virulence Variation in Cochliobolus sativus in China

Spot blotch caused by Cochliobolus sativus has become an important disease in the wheat-growing regions in China that has resulted from changes in the regional climate, agricultural cultivation patterns, and the susceptible wheat varieties that are widely grown. Little information is available about virulence variability and pathogenic specialization of the C. sativus isolates from major wheat-growing regions in China. Here, 12 representative wheat varieties and foundation breeding stocks were selected to characterize the pathotypes of C. sativus isolates from infected wheat plants. Based on the infection phenotypes in the 12 differential genotypes at the seedling stage, 70 Chinese pathotypes were identified from 110 isolates and clustered into three virulence groups. The high virulence isolates were collected from wheat leaves, crowns, and roots, with most (10 of 14) from the Henan province in the Huang-Huai plain. No relationship was evident between virulence variability of C. sativus isolates and their geographic origins or types of diseased wheat tissues. C. sativus showed a significant pathogenic specialization in hosts of wheat and barley. Most of the wheat isolates (50 of 65) were avirulent to all the differential barley genotypes, and a few were virulent only to highly susceptible barley genotypes. These results indicated that C. sativus isolates from the wheat-growing regions in China varied considerably for their virulence in wheat varieties, and showed significant pathogenic specialization to the wheat and barley hosts.

SSR MARKERS REVEALED GENETIC DIVERGENCE OF RICE BROWN PLANTHOPPER POPULATIONS MAINTAINED ON TWO SETS OF DIFFERENTIAL HOST VARIETIES

<p>Resistance screening of promising rice lines in Indonesia requires the use of brown planthopper (BPH) biotypes 1, 2, and 3. Three BPH populations have been raised as biotypes 1, 2, and 3 on differential rice host of improved varieties Pelita I-1 (no <em>Bph </em>gene), IR26 (<em>Bph1</em>), and IR42 (<em>bph2</em>), respectively. Three alternative populations have also been developed on the respective traditional varieties TN1 (no <em>Bph </em>gene), Mudgo (<em>Bph1</em>), and ASD7 (<em>bph2</em>). Although these populations displayed two virulent patterns other than biotype 1 to 3 phenotypes, they were expected to be discriminated into two virulence groups by SSR analysis. The study aimed to investigate the level of genetic variation among the six BPH populations using SSR markers and to relate it with the observed virulence patterns. Genotyping of 30 females with 29 polymorphic SSR markers revealed higher genetic parameter values in populations reared on improved varieties than those on traditional varieties. This difference was marked as two population clusters in PCoA plots corresponding to the host variety type, in contrast to the previous assumption that clustering would be based on virulence patterns. The presence of individuals with unwanted virulence allele, either resulting from contamination during the long period of rearing or lack of host adaptation period, is suspected. The result of this study indicates that the six populations are not suitable for resistance screening. Virulence selection must be performed until they attain biotype 1 to 3 phenotypes which can be genetically separated by DNA markers.</p>

Genomic Characterization of Streptococcus suis Serotype 24 Clonal Complex 221/234 From Human Patients.

Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. Although S. suis serotype 2 is prevalent among patient and swine infections, other serotypes are occasionally detected in humans. Of these, serotype 24 clonal complex (CC) 221/234 are recognized as emerging clones of human infection. Genomic exploration of three S. suis serotype 24 CC221/234 strains revealed antimicrobial resistance genes, pathotyping, virulence-associated gene (VAG) profiles, minimum core genome (MCG) typing, and comparison of the genomes. Based on these analyzes, all three serotype 24 strains were MCG7-3 and should be classified in the intermediate/weakly virulent (I/WV) group. All selected serotype 24 strains were susceptible to several antibiotics including β-lactam, fluoroquinolone, and chloramphenicol. Resistance to tetracycline, macrolide, and clindamycin was observed and attributed to the genes tet(O) and erm(B). Genomic comparison revealed the strains S12X, LSS66, LS0L, LS0E, 92–4,172, and IMT40201 that had phylogenetic affinity with serotype 24 CC221/234. Analysis of 80 virulence-associated genes (VAG) showed that all three serotype 24 strains lacked 24 genes consisting of adhesin P, epf, hyl, ihk, irr, mrp, nadR, neuB, NisK/R, ofs, permease (SSU0835), rgg, revS, salK/R, sao, sly, spyM3_0908, srtBCD, srtF, srtG, SSU05_0473, virA, virB4, and virD4. Eleven specific sequences were identified in the 3 serotype 24 genomes that differed from the genomes of the representative strains of epidemic (E; SC84), highly virulent (HV; P1/7), I/WV (89–1,591), and avirulent (T15 and 05HAS68).

746. Clinical and Microbiological Characteristics of Clostridioides difficile Infection After a Change in Diagnostic Testing Algorithm

Abstract Background There is a lack of consensus on the most appropriate testing for C. difficile infection (CDI). The objective of this study was to determine the clinical and microbiological characteristics of CDI following a switch from stool PCR testing only to PCR reflexed to toxin testing. Figure 1. PCR Cycle Threshold Values Methods We reviewed the characteristics and outcomes of 50 consecutive patients who tested positive for CD by PCR (Xpert CD, Cepheid) between October 2019 and January 2020 at the Hines VA Hospital. Cases were defined by results of reflex toxin testing (Cdiff quick check complete, Alere/TechLab) after a positive PCR result. Baseline characteristics, symptoms, initial laboratory data, and treatments were compared as well as patient outcomes, including hospital readmission due to CDI at 30 days, and recurrent CDI (rCDI) at 30 and 90 days. The cycle threshold for the stool PCR result was recorded. Stools were cultured anaerobically for CD, and restriction endonuclease analysis (REA) strain typing was performed on the recovered CD isolates. Results Toxin testing was positive in 19/50 (38%) cases. Compared to stool toxin-negative cases, toxin-positive cases were older (95% vs. 71% were age ≥ 65, p = 0.06), more likely to have a history of CDI (37% vs. 23%, p = 0.34), and have ≥ 1 CDI episodes within 6 months (37% vs. 19%, p = 0.26). Treatment for CDI was more common in patients who had a positive toxin text. (95% vs 61%, p= 0.009). Among the 38 patients that received treatment, 33 received vancomycin (87%) and 8 patients (21%) had rCDI at 30 days. Of the 8 patients with rCDI, 2 were re-admitted to the hospital for CDI. The average PCR cycle threshold was lower in the toxin-positive stools compared to toxin-negative stools (24.46 and 29.96, p< 0.001; Fig. 1) The endemic REA group Y was the most common CD strain recovered (30%) and the previously epidemic and virulent REA group BI strain was recovered in 11% of the cases. Conclusion CDI cases diagnosed by positive stool PCR and positive toxin tests had more typical risk factors for CDI, a lower PCR cycle threshold and were more likely to have been treated for CDI. Outcomes were similar in this setting where infection with the virulent BI strain was uncommon. Disclosures Stuart Johnson, MD, Acurx Pharmaceuticals (Advisor or Review Panel member)Bio-K+ (Advisor or Review Panel member)Ferring Pharmaceutical (Advisor or Review Panel member)

Races of Puccinia striiformis f. sp. tritici identified from the coastal areas of Turkey

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a devastating disease of wheat in Turkey and worldwide. This pathogen can overcome known resistance genes and negatively affect the wheat production. The objectives of this study were to determine the virulence patterns of wheat stripe rust isolates collected from the coastal areas (Aegean and Mediterranean regions) of Turkey, and to examine the genetic diversity of the pathogen populations by simple sequence repeat (SSR) marker analysis. The majority of the isolates collected were virulent at various frequencies on the resistance genes Yr1 (50%), Yr6 (100%), Yr7 (78%), Yr8 (50%), Yr9 (84%), Yr10 (25%), Yr17 (38%), Yr24 (22%), Yr27 (31%), Yr32 (22%), Yr43 (47%), Yr44 (6%), YrSp (41%), YrTr1 (6%), and avirulent on Yr5 and Yr15. Based on the analysis of virulence, all isolates were determined as 25 races and clustered into six virulence groups (VGs). In contrast, the isolates clustered into four molecular groups (MGs) based on genotypic data. All four MGs were found in Aegean region of Turkey, while only three MGs (MG1, MG2 and MG3) were found in the Mediterranean region of the country. Additionally, analysis of molecular variance indicated that 80.9% of genetic variation was found within regions and 19.1% was found between the regions. The results suggest that interregional migration of the pathogen was high. The data can be helpful for the management of stripe rust and understanding the population structure and migration of Pst in Turkey.

Virulence characterization of Puccinia striiformis f. sp. tritici collections from six countries in 2013 to 2020

Puccinia striiformis f. sp. tritici (Pst) causes wheat stripe rust (also called yellow rust, Yr), one of the most important diseases worldwide. Characterization of virulence in Pst populations is essential for developing wheat cultivars with effective and durable resistance to control the disease. A total of 138 Pst races, including 120 races that were not previously reported, were identified from stripe rust collections made from Canada, China, Ecuador, Egypt, Italy and Mexico in 2013–2020 using a set of 18 Yr single-gene differentials. Virulence of the resistance gene Yr5 or Yr15 was not found in isolates from any of the countries, indicating their effectiveness against the Pst populations. Virulence to 16 Yr genes was detected, but the frequencies varied greatly among countries. On average, the frequencies of virulence to Yr6, Yr7, Yr9, Yr43, Yr44 and YrExp2 were high (81.7–90.6%), those to Yr1, Yr8, Yr17, Yr27, YrSP and Yr76 were moderate (34.0–56.8%), and those to Yr10, Yr24, Yr32 and YrTr1 were low or very low (0.4–18.5%). The same races detected in different countries and different races from different countries clustered into the same virulence groups, indicating Pst migration among different countries, especially between eastern Asia and the Mediterranean region. These results should be useful for breeding wheat cultivars with effective resistance to stripe rust in these countries as well as globally.

SRAP analysis of brown planthopper (Nilaparvata lugens) populations maintained on differential rice host varieties

Abstract. Chaerani, Dadang A, Fatimah, Husin BA, Sutrisno, Yunus M. 2021. SRAP analysis of brown planthopper (Nilaparvata lugens) populations maintained on differential rice host varieties. Biodiversitas 22: 4266-4272. Brown planthopper (Nilaparvata lugens Stål) biotypes differ in virulence to rice varieties carrying different Bph resistance genes. These biotypes are reported can be genetically discriminated against using DNA markers. Four brown planthoppers (BPH) populations, which displayed two virulence phenotypes, have been produced by selection and adaptation on four differential host varieties. Sequence-related amplified polymorphism (SRAP) marker preferentially amplifies the coding regions in the genome and, thus, can discriminate the observed virulence variations among those populations. This study aimed to analyze the genetic variation of four developed BPH populations using SRAP markers. Genetic analysis of a total of 40 BPH females with 18 polymorphic primers revealed equal genetic diversity parameter values among populations (Na: 1.1 to 1.4, Ne: 1.2 to 1.3, I: 0.22 to 0.29, He: 0.14 to 0.18, and UHe: 0.15 to 0.19). Analysis of population structure by AMOVA indicated low genetic variation among populations (9%). Still, pairwise PhiPT population values between all pairs of the population revealed the presence of moderate genetic differentiations (PhiPT ranged from 0.57 to 0.133, P<0.01). Two partial clusters in plots of PCoA were corresponded to two virulence groups, indicating the ability of SRAP markers to discriminate virulence phenotype. Further selection and adaptation are expected can form four desired virulence patterns with complete genetic separation among the population before its application as resistance screening agents of rice lines.

Transcriptomic profiling reveals different innate immune responses in primary alveolar macrophages infected by two highly homologous porcine reproductive and respiratory syndrome viruses with distinct virulence

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates show high genetic and pathogenic diversity. The mechanisms underlying different virulence of PRRSV isolates are still not fully clarified. Two highly homologous PRRSV isolates (XJ17-5 and JSTZ1712-12) with distinct virulence were identified in our previous study. To evaluate the association between host responses and different virulence, here we investigated the transcriptomic profiles of porcine alveolar macrophages (PAMs) infected with these two isolates. RNA-Seq results showed that there are 1932 differential expression genes (DEGs) between two PRRSV infected groups containing 1067 upregulation and 865 downregulation genes. Compared with the avirulent JSTZ1712-12 infected group, GO analysis identified significant enrichment gene sets not only associated with virus infection but also innate immune response in the virulent XJ17-5 infected group. In addition, KEGG analysis indicated significantly enriched genes associated with NOD-like and RIG-I-like receptor signaling pathways in XJ17-5 vs JSTZ1712-12 group. Furthermore, XJ17-5 isolate induced significantly higher levels of innate immune response associated genes (IL-1β, CXCL2, S100A8, OAS2, MX1, IFITM3, ISG15 and IFI6) than JSTZ1712-12 isolate, which were further confirmed by real-time PCR. Given that these two isolates share similar replication efficiency in vivo and in vitro, our results indicated that distinct virulence of PRRSV isolates is associated with different host innate immune responses.

The Role of the Respiratory Microbiome and Viral Presence in Lower Respiratory Tract Infection Severity in the First Five Years of Life.

Lower respiratory tract infections (LRTIs) in children are common and, although often mild, a major cause of mortality and hospitalization. Recently, the respiratory microbiome has been associated with both susceptibility and severity of LRTI. In this current study, we combined respiratory microbiome, viral, and clinical data to find associations with the severity of LRTI. Nasopharyngeal aspirates of children aged one month to five years included in the STRAP study (Study to Reduce Antibiotic prescription in childhood Pneumonia), who presented at the emergency department (ED) with fever and cough or dyspnea, were sequenced with nanopore 16S-rRNA gene sequencing and subsequently analyzed with hierarchical clustering to identify respiratory microbiome profiles. Samples were also tested using a panel of 15 respiratory viruses and Mycoplasma pneumoniae, which were analyzed in two groups, according to their reported virulence. The primary outcome was hospitalization, as measure of disease severity. Nasopharyngeal samples were isolated from a total of 167 children. After quality filtering, microbiome results were available for 54 children and virology panels for 158 children. Six distinct genus-dominant microbiome profiles were identified, with Haemophilus-, Moraxella-, and Streptococcus-dominant profiles being the most prevalent. However, these profiles were not found to be significantly associated with hospitalization. At least one virus was detected in 139 (88%) children, of whom 32.4% had co-infections with multiple viruses. Viral co-infections were common for adenovirus, bocavirus, and enterovirus, and uncommon for human metapneumovirus (hMPV) and influenza A virus. The detection of enteroviruses was negatively associated with hospitalization. Virulence groups were not significantly associated with hospitalization. Our data underlines high detection rates and co-infection of viruses in children with respiratory symptoms and confirms the predominant presence of Haemophilus-, Streptococcus-, and Moraxella-dominant profiles in a symptomatic pediatric population at the ED. However, we could not assess significant associations between microbiome profiles and disease severity measures.

Zoonotic vaccinia virus strains belonging to different genetic clades exhibit immunomodulation abilities that are proportional to their virulence

BackgroundThe vaccinia virus (VACV) isolates, Guarani P1 virus (GP1V) and Passatempo virus (PSTV), were isolated during zoonotic outbreaks in Brazil. Each one of them belongs to two different VACV clades, defined by biological aspects that include virulence in mice and phylogenetic analysis. Considering that information about how vaccinia viruses from different groups elicit immune responses in animals is scarce, we investigated such responses in mice infected either by GP1V (group 2) or PSTV (group 1), using VACV Western Reserve strain (VACV-WR) as control.MethodsThe severity of the infections was evaluated in BALB/c mice considering diverse clinical signs and defined scores, and the immune responses triggered by GP1V and PSTV infections were analysed by immune cell phenotyping and intra-cytoplasmic cytokines detection.ResultsWe detected a reduction in total lymphocytes (CD3 +), macrophages (CD14 +), and NK cells (CD3-CD49 +) in animals infected with VACV-WR or GP1V. The VACV-WR and GP1V viruses, belonging to the most virulent group in a murine model, were able to down-modulate the cell immune responses upon mice infection. In contrast, PSTV, a virus considered less virulent in a murine model, showed little ability to down-modulate the mice immune responses. Mice infected with VACV-WR and GP1V viruses presented significant weight loss and developed lesions in their spleens, as well as damage to liver and lungs whereas mice infected with PSTV developed only moderate clinical signs.ConclusionsOur results suggest that VACV immunomodulation in vivo is clade-related and is proportional to the strain’s virulence upon infection. Our data corroborate the classification of the different Brazilian VACV isolates into clades 1 and 2, taking into account not only phylogenetic criteria, but also clinical and immunological data.

Comparative Secretome and Functional Analyses Reveal Glycoside Hydrolase Family 30 and Cysteine Peptidase as Virulence Determinants in the Pinewood Nematode Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pinewood nematode, Bursaphelenchus xylophilus, is one of the world’s most serious tree diseases. Although the B. xylophilus whole-genome sequence and comprehensive secretome profile have been determined over the past decade, it remains unclear what molecules are critical in pine wilt disease and govern B. xylophilus virulence in host pine trees. Here, a comparative secretome analysis among four isolates of B. xylophilus with distinct virulence levels was performed to identify virulence determinants. The four candidate virulence determinants of B. xylophilus highly secreted in virulent isolates included lipase (Bx-lip1), glycoside hydrolase family 30 (Bx-GH30), and two C1A family cysteine peptidases (Bx-CAT1 and Bx-CAT2). To validate the quantitative differences in the four potential virulence determinants among virulence groups at the protein level, we used real-time reverse-transcription polymerase chain reaction analysis to investigate these determinants at the transcript level at three time points: pre-inoculation, 3 days after inoculation (dai), and 7 dai into pine seedlings. The transcript levels of Bx-CAT1, Bx-CAT2, and Bx-GH30 were significantly higher in virulent isolates than in avirulent isolates at pre-inoculation and 3 dai. A subsequent leaf-disk assay based on transient overexpression in Nicotiana benthamiana revealed that the GH30 candidate virulent factor caused cell death in the plant. Furthermore, we demonstrated that Bx-CAT2 was involved in nutrient uptake for fungal feeding via soaking-mediated RNA interference. These findings indicate that the secreted proteins Bx-GH30 and Bx-CAT2 contribute to B. xylophilus virulence in host pine trees and may be involved in pine wilt disease.

Diversity and Virulence of Streptomyces spp. Causing Potato Common Scab in Prince Edward Island, Canada.

Common scab (CS) is a potato disease that significantly decreases the market value of potato tubers after the development of necrotic lesions on their surface. Streptomyces scabiei is the main causal agent of CS; however, other closely related species, including S. acidiscabies and S. turgidiscabies, have also been shown to cause the disease. In this study, we characterized the genetic and phenotypic diversity of Streptomyces spp. causing CS in Prince Edward Island, the main potato-producing province in Canada. Two hundred and ninety-six pathogenic Streptomyces spp. isolates were retrieved from diseased tubers harvested from six fields located across a longitudinal geographical gradient. Genome fingerprinting analyses using repetitive elements PCR (ERIC- and BOX-PCR) revealed 14 distinct genetic groups. Thirteen groups were taxonomically affiliated with S. scabiei, whereas the fourteenth group was affiliated with S. acidiscabies. Their geographical distribution was characterized and revealed that on average between six and eight different genetic groups were detected per field, with variable abundance. Virulence assays showed strong differences in virulence between the genetic groups, ranging from low to highly virulent. Interestingly, pathogenic Streptomyces spp. populations in each field seem to be dominated by the most virulent genetic groups. The results obtained will contribute to better understanding of the population dynamic of pathogenic Streptomyces spp. causing CS of potato and promoting the development of more efficient detection and intervention tools to manage this important potato disease.