Identification Of Virulence Genes Research Articles

ZymoSoups: A High-Throughput Forward Genetics Method for Rapid Identification of Virulence Genes in Zymoseptoria tritici.

Septoria tritici blotch is caused by the fungus Zymoseptoria tritici and poses a major threat to wheat productivity. There are over 20 mapped loci in wheat that confer strong (gene-for-gene) resistance against this pathogen, but the corresponding genes in Z. tritici that confer virulence against distinct R genes remain largely unknown. In this study, we developed a rapid forward genetics methodology to identify genes that enable Z. tritici to gain virulence on previously resistant wheat varieties. We used the known gene-for-gene interaction between Stb6 and AvrStb6 as a proof of concept that this method could quickly recover single candidate virulence genes. We subjected the avirulent Z. tritici strain IPO323, which carries the recognized AvrStb6 allele, to ultraviolet (UV) mutagenesis and generated a library of over 66,000 surviving spores. We screened these survivors on leaves of the resistant wheat variety Cadenza in mixtures (soups) ranging from 100 to 500 survivors per soup. We identified five soups with a gain-of-virulence (GoV) phenotype relative to the IPO323 parental strain and re-sequenced 18 individual isolates, including four control isolates and two isolates lacking virulence, when screened individually. Of the 12 confirmed GoV isolates, one had a single nucleotide polymorphism (SNP) in the AvrStb6 coding region. The other 11 GoV isolates exhibited large (approximately 70 kb) deletions at the end of chromosome 5, including the AvrStb6 locus. Our findings demonstrate the efficiency of this forward genetic approach in elucidating the genetic basis of qualitative resistance to Z. tritici and the potential to rapidly identify other, currently unknown, Avr genes in this pathogen. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification of virulence genes from clinical isolates of Methicillin-Resistant Staphylococcus aureus (MRSA)

Abstract. Mohammad SQ, Awayid HS, Zarrouk-Mahjoub S. 2024. Identification of virulence genes from clinical isolates of Methicillin-Resistant Staphylococcus aureus (MRSA). Biodiversitas 25: 5083-5093. Methicillin-resistant Staphylococcus aureus (MRSA) is a strain of Staphylococcus aureus that has developed resistance to beta-lactam antibiotics, including penicillin, methicillin, amoxicillin, and oxacillin. This resistance complicates treatment compared to non-resistant strains. While previous studies have defined MRSA phylogeny and transmission in well-resourced settings, this study specifically highlights the effectiveness of five targeted loci analyses (ermA, ermB, ermC, hla-?, and tsst-1) for understanding MRSA evolution in diverse clinical contexts. This study analyzed the phylogenetic relationships among 20 MRSA isolates, which were amplified from the targeted loci. The research focused on the ermA locus in three isolates (A1-A3), the ermB locus in nine isolates (B1 to B9), the ermC locus in six isolates (C1 to C6), the hla-? gene in one isolate (D1), and the tsst-1 gene in one isolate (E1). Genetic diversity was assessed based on the variants identified within these loci. Direct sequencing of the amplified fragments was conducted to detect genetic polymorphisms, and coding variants were translated to evaluate their potential effects on protein function. A phylogenetic tree was constructed to evaluate the associations and distributions of the experimental variables. Sequencing results confirmed that the isolates were S. aureus. The alignment revealed no variants in samples A1-A3, C1-C6, and D1. One variant (176G>A) was found in group B1-B9, resulting in a missense mutation (p.100Ser>Asn). Sample E1 contained a single nucleotide deletion (56C-del). Phylogenetic analysis revealed distinct clades of isolates that corresponded with various clinical and non-clinical sources. The ermA locus proved to be more effective in tracking the evolutionary trajectories of the studied S. aureus isolates compared to the other loci. These findings suggest that ermA markers could be valuable for broader applications in monitoring the evolutionary distributions of bacterial strains across diverse contexts. Further investigation may enhance our understanding of bacterial evolution in various environments.

Exploring phenotypic and genotypic diversity among methicillin-resistant, vancomycin-resistant, and sensitive Staphylococcus aureus

Background: Methicillin-Resistant Staphylococcus aureus (MRSA) is a global concern owing to the increasing prevalence of multidrug-resistant (MDR) strains. Vancomycin has been the primary treatment for MRSA; however, Vancomycin-resistant strains are being increasingly reported worldwide. Therefore, comparative studies are essential to support antimicrobial stewardship and improving clinical management. Ultimately, the findings from this study are expected to inform treatment strategies and guide public health interventions effectively. Material and methods: This study investigated the prevalence, antimicrobial resistance, and virulence characteristics of Vancomycin-sensitive S. aureus (VSSA) and Vancomycin-resistant S. aureus (VRSA) within MRSA strains. By employing a combination of phenotypic methods, such as antimicrobial susceptibility testing, and genotypic techniques, including molecular typing and identification of virulence genes, we obtained comprehensive insights into VRSA and VSSA profiles. Results: Of 250 clinical samples, 62 (24.8%) were S. aureus and 27 (43.5%) were identified as MRSA. All MRSA isolates exhibited MDR patterns. Most MRSA strains were VSSA (20/27, 74.1%), while 7 (25.9%) were VRSA. The VRSA isolates showed more antimicrobial resistance than VSSA isolates; however, the VRSA isolates had less virulence than VSSA isolates. Linezolid was the most effective treatment, with a 3.7% resistance rate. A higher percentage of biofilm-producing MRSA (96.3%) was confirmed by both phenotypic and genotypic methods. All isolates, except one VRSA, showed multi-virulence patterns (harbored more than 3 virulence genes). High diversity and low clonality (D-value = 0.99) were found in both VSSA and VRSA. Based on our correlation findings, the emergence of vancomycin resistance could modify the association between antimicrobial resistance and virulence, potentially affecting the pathogenic profile of these strains. The study also revealed complex interactions among host factors (including age and gender), sample origin, antimicrobial resistance, biofilm production, and virulence genes. Conclusion: This study highlights the alarming spread of MRSA and VRSA, which show significant resistance and virulence.

Exploring probiotic potential and antimicrobial properties of lactic acid bacteria from cow's milk

Exploring probiotic potential and antimicrobial properties of lactic acid bacteria from cow's milk

Whole Genome Analysis of Tibetan Kefir-Derived Lactiplantibacillus Plantarum 12-3 Elucidates Its Genomic Architecture, Antimicrobial and Drug Resistance, Potential Probiotic Functionality and Safety.

Lactiplantibacillus plantarum 12-3 holds great promise as a probiotic bacterial strain, yet its full potential remains untapped. This study aimed to better understand this potential therapeutic strain by exploring its genomic landscape, genetic diversity, CRISPR-Cas mechanism, genotype, and mechanistic perspectives for probiotic functionality and safety applications. L. plantarum 12-3 was isolated from Tibetan kefir grains and, subsequently, Illumina and Single Molecule Real-Time (SMRT) technologies were used to extract and sequence genomic DNA from this organism. After performing pan-genomic and phylogenetic analysis, Average Nucleotide Identity (ANI) was used to confirm the taxonomic identity of the strain. Antibiotic resistance gene analysis was conducted using the Comprehensive Antibiotic Resistance Database (CARD). Antimicrobial susceptibility testing, and virulence gene identification were also included in our genomic analysis to evaluate food safety. Prophage, genomic islands, insertion sequences, and CRISPR-Cas sequence analyses were also carried out to gain insight into genetic components and defensive mechanisms within the bacterial genome. The 3.4 Mb genome of L. plantarum 12-3, was assembled with 99.1% completeness and low contamination. A total of 3234 genes with normal length and intergenic spacing were found using gene prediction tools. Pan-genomic studies demonstrated gene diversity and provided functional annotation, whereas phylogenetic analysis verified taxonomic identity. Our food safety study revealed a profile of antibiotic resistance that is favorable for use as a probiotic. Analysis of insertional sequences, genomic islands, and prophage within the genome provided information regarding genetic components and their possible effects on evolution. Pivotal genetic elements uncovered in this study play a crucial role in bacterial defense mechanisms and offer intriguing prospects for future genome engineering efforts. Moreover, our findings suggest further in vitro and in vivo studies are warranted to validate the functional attributes and probiotic potential of L. plantarum 12-3. Expanding the scope of the research to encompass a broader range of L. plantarum 12-3 strains and comparative analyses with other probiotic species would enhance our understanding of this organism's genetic diversity and functional properties.

A Hydroponic-Based Bioassay to Facilitate Plasmodiophora brassicae Phenotyping.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases affecting the canola/oilseed rape (Brassica napus) industry worldwide. Currently, the planting of clubroot-resistant (CR) cultivars is the most effective strategy used to restrict the spread and the economic losses linked to the disease. However, virulent P. brassicae isolates have been able to infect many of the currently available CR cultivars, and the options to manage the disease are becoming limited. Another challenge has been achieving consistency in evaluating host reactions to P. brassicae infection, with most bioassays conducted in soil and/or potting medium, which requires significant space and can be labor intensive. Visual scoring of clubroot symptom development can also be influenced by user bias. Here, we have developed a hydroponic bioassay using well-characterized P. brassicae single-spore isolates representative of clubroot virulence in Canada, as well as field isolates from three Canadian provinces in combination with canola inbred homozygous lines carrying resistance genetics representative of CR cultivars available to growers in Canada. To improve the efficiency and consistency of disease assessment, symptom severity scores were compared with clubroot evaluations based on the scanned root area. According to the results, this bioassay offers a reliable, less expensive, and reproducible option to evaluate P. brassicae virulence, as well as to identify which canola resistance profile(s) may be effective against particular isolates. This bioassay will contribute to the breeding of new CR canola cultivars and the identification of virulence genes in P. brassicae that could trigger resistance and that have been very elusive to this day.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Whole genome sequencing in the palm of your hand: how to implement a MinION Galaxy-based workflow in a food safety laboratory for rapid Salmonella spp. serotyping, virulence, and antimicrobial resistance gene identification.

Whole Genome Sequencing (WGS) implementation in food safety laboratories is a significant advancement in food pathogen control and outbreak tracking. However, the initial investment for acquiring next-generation sequencing platforms and the need for bioinformatic skills represented an obstacle for the widespread use of WGS. Long-reading technologies, such as the one developed by Oxford Nanopore Technologies, can be easily implemented with a minor initial investment and with simple protocols that can be performed with basic laboratory equipment. Herein, we report a simple MinION Galaxy-based workflow with analysis parameters that allow its implementation in food safety laboratories with limited computer resources and without previous knowledge in bioinformatics for rapid Salmonella serotyping, virulence, and identification of antimicrobial resistance genes. For that purpose, the single use Flongle flow cells, along with the MinION Mk1B for WGS, and the community-driven web-based analysis platform Galaxy for bioinformatic analysis was used. Three strains belonging to three different serotypes, monophasic S. Typhimurium, S. Grancanaria, and S. Senftenberg, were sequenced. After 24 h of sequencing, enough coverage was achieved in order to perform de novo assembly in all three strains. After evaluating different tools, Flye de novo assemblies with medaka polishing were shown to be optimal for in silico Salmonella spp. serotyping with SISRT tool followed by antimicrobial and virulence gene identification with ABRicate. The implementation of the present workflow in food safety laboratories with limited computer resources allows a rapid characterization of Salmonella spp. isolates.

Identification of potential pathogenic targets and survival strategies of Vibrio vulnificus through population genomics.

Vibrio vulnificus, a foodborne pathogen, has a high mortality rate. Despite its relevance to public health, the identification of virulence genes associated with the pathogenicity of currently known clinical isolates of V. vulnificus is incomplete and its synergistic pathogenesis remains unclear. Here, we integrate whole genome sequencing (WGS), genome-wide association studies (GWAS), and genome-wide epistasis studies (GWES), along with phenotype characterization to investigate the pathogenesis and survival strategies of V. vulnificus. GWAS and GWES identified a total of six genes (purH, gmr, yiaV, dsbD, ramA, and wbpA) associated with the pathogenicity of clinical isolates related to nucleotide/amino acid transport and metabolism, cell membrane biogenesis, signal transduction mechanisms, and protein turnover. Of these, five were newly discovered potential specific virulence genes of V. vulnificus in this study. Furthermore, GWES combined with phenotype experiments indicated that V. vulnificus isolates were clustered into two ecological groups (EGs) that shared distinct biotic and abiotic factors, and ecological strategies. Our study reveals pathogenic mechanisms and their evolution in V. vulnificus to provide a solid foundation for designing new vaccines and therapeutic targets.

Identification of Antimicrobial Resistance in Faecal Microbes from Wild Dugongs (Dugong dugon)

Estuarine and coastal waters are areas of potential concern for antimicrobial resistance because of the discharge of wastewater from sewage treatment plants and the run-off from urban and agricultural lands. Herein, we evaluate the antimicrobial resistance profiles in bacteria from dugongs (Dugong dugon), mammals that inhabit and feed in shallow coastal regions and, thus, are vulnerable to encountering water and sediment contaminated by human activities. Bacterial isolates were cultured from fresh faeces of four wild dugongs, as well as from one sediment sample from a dugong foraging ground in Queensland, Australia. Ten bacterial isolates underwent phenotypic antimicrobial susceptibility testing using disc diffusion and minimum inhibitory concentration testing, and genotypic resistance and virulence gene identification through whole genome sequencing. Four Staphylococcus warneri isolates and one Bacillus cereus isolate from dugong faeces were resistant to penicillin, with two S. warneri isolates also displaying resistance to trimethoprim. Four Escherichia coli isolates were all resistant to ampicillin. Resistance genes, including fosB, BcII, dfrC, blaZ, and mdfA, were identified in the isolates cultured from dugong faeces with two virulence genes (gad and lpfA) identified in all E. coli isolates. Lysinibacillus sphaericus cultured from the marine sediment and B. cereus from dugong faeces displayed phenotypic multidrug resistance (across categories of non-extended spectrum cephalosporins, penicillins and beta-lactamase inhibitors, and clindamycin; and for L. sphaericus, phosphonic acids). These results demonstrate the role that dugongs can play as a sentinel species for antimicrobial resistance in the coastal waters across their range, which includes both disturbed urban and rural regions.

Virulence, antimicrobial susceptibility and phylogenetic analysis of Cronobacter sakazakii isolates of food origins from Jordan.

The aim was to characterize a collection of Cronobacter sakazakii isolates collected from various origins in Jordan. The isolates were characterized using 16S rRNA sequencing, DNA microarray, multi-locus sequence typing (MLST), O-serotyping, virulence gene identification and antibiotic susceptibility testing. The identities and phylogenetic relatedness revealed that C. sakazakii sequence type 4 (ST4) and Csak O:1 serotype were the most prevalent STs and serovars amongst these C. sakazakii strains. PCR screening of putative virulence genes showed that the siderophore-interacting protein gene (sip) and iron acquisition gene clusters (eitCBAD and iucABCD/iutA) were the most detected genes with noticeable variability in the type 6 secretion system (T6SS) and filamentous hemagglutinin/adhesion (FHA) gene loci. The antibiotic resistance profiles revealed that the majority of the isolates were susceptible to all antibiotics used despite harbouring a class C β-lactamase resistance gene. The results described in this report provide additional insights about the considerable genotypic and phenotypic heterogeneity within C. sakazakii. The information reported in this study might be of great value in understanding the origins of C. sakazakii isolates, in addition to their diversity and variability, which might be helpful in preventing future outbreaks of this pathogen.

Isolation of ferric Yersinia bactin A (fyuA) as virulence gene and biofilm forming in Escherichia coli was Collected from patient with UTI.

The focus of the current investigation was applied on determining the presence of ferric yersiniabactin uptake A (fyuA) virulence gene and identifying the antibiotic resistance properties in biofilm forming Escherichia coli­­ isolated from recurrent urinary tract infection patients and healthy individuals. In the Microbiology laboratory at Al Yarmouk hospital in Baghdad, Iraq, 20 (16 females and 4 males) UTI-diagnosed patient urine and stool samples, as well as 20 (10 females and 10 males) healthy individual stool samples, were collected. The samples were exposure to series test including O and H antigen based identification using an antibody (polyvalent and monovalent)-directed agglutination test, antibiotic sensitivity testing using the Kirby Bauer disc diffusion method, Congo-red agar method-based biofilm formation identification, and virulence gene identification using polymerase chain reaction (PCR).Results: 40 and 20 strains were discovered from UTI patients and healthy persons, respectively, in which O serogroups were found in 7(35%) and 3(15%) of those strains, respectively. Furthermore, only two strains of enterohemorrhagic E. coli (O157 H7) and enteroaggregative E. coli (O44 H7) were found in the UTI-based urine samples. Multidrug resistance (MDR) was detected in 60% of the biofilm positive producers (BPPs) isolated from UTI urine samples; however, only 20% of the biofilm negative producers (BNPs) isolated from UTI urine samples displayed MDR features. In the instance of virulence genes, a link was shown between fyuA (a gene that codes for an outer-membrane protein called ferric yersiniabactin uptake) and biofilm in 75% and 60% of UTI urine and stool strains, respectively, and in 55% of healthy individual stool strains. The findings of the antibiotic sensitivity test revealed that the UTI strains were 90 percent, 100 percent, 65 percent, and 40 percent sensitive to imipenem, amikacin, gentamicin, and tobramycin, respectively. Conclusion:The present research offers detailed information on the biofilm, virulence gene, and antibiotic resistance features of Escherichia coli from recurrent urinary tract infection patients and healthy people.

Hcp Proteins of the Type VI Secretion System Promote Avian Pathogenic E. coli DE205B (O2:K1) to Induce Meningitis in Rats.

Avian pathogenic Escherichia coli (APEC) is an important extra-intestinal pathogenic E. coli (ExPEC), which often causes systemic infection in poultry and causes great economic loss to the breeding industry. In addition, as a major source of human ExPEC infection, the potential zoonotic risk of APEC has been an ongoing concern. Previous studies have pointed out that APEC is a potential zoonotic pathogen, which has high homology with human pathogenic E. coli such as uro-pathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC), shares multiple virulence factors and can cause mammalian diseases. Previous studies have reported that O18 and O78 could cause different degrees of meningitis in neonatal rats, and different serotypes had different degrees of zoonotic risk. Here, we compared APEC DE205B (O2:K1) with NMEC RS218 (O18:K1:H7) by phylogenetic analysis and virulence gene identification to analyze the potential risk of DE205B in zoonotic diseases. We found that DE205B possessed a variety of virulence factors associated with meningitis and, through phylogenetic analysis, had high homology with RS218. DE205B could colonize the cerebrospinal fluid (CSF) of rats, and cause meningitis and nerve damage. Symptoms and pathological changes in the brain were similar to RS218. In addition, we found that DE205B had a complete T6SS, of which Hcp protein was its important structural protein. Hcp1 induced cytoskeleton rearrangement in human brain microvascular endothelial cells (HBMECs), and Hcp2 was mainly involved in the invasion of DE205B in vitro. In the meningitis model of rats, deletion of hcp2 gene reduced survival in the blood and the brain invasiveness of DE205B. Compared with WT group, Δhcp2 group induced lower inflammation and neutrophils infiltration in brain tissue, alleviating the process of meningitis. Together, these results suggested that APEC DE205B had close genetic similarities to NMEC RS218, and a similar mechanism in causing meningitis and being a risk for zoonosis. This APEC serotype provided a basis for zoonotic research.

The In Vitro Replication Cycle of Achromobacter xylosoxidans and Identification of Virulence Genes Associated with Cytotoxicity in Macrophages.

ABSTRACTAchromobacter xylosoxidans is an opportunistic pathogen implicated in a wide variety of human infections including the ability to colonize the lungs of cystic fibrosis (CF) patients. The role of A. xylosoxidans in human pathology remains controversial due to the lack of optimized in vitro and in vivo model systems to identify and test bacterial gene products that promote a pathological response. We have previously identified macrophages as a target host cell for A. xylosoxidans-induced cytotoxicity. By optimizing our macrophage infection model, we determined that A. xylosoxidans enters macrophages and can reside within a membrane bound vacuole for extended periods of time. Intracellular replication appears limited with cellular lysis preceding an enhanced, mainly extracellular replication cycle. Using our optimized in vitro model system along with transposon mutagenesis, we identified 163 genes that contribute to macrophage cytotoxicity. From this list, we characterized a giant RTX adhesin encoded downstream of a type one secretion system (T1SS) that mediates bacterial binding and entry into host macrophages, an important first step toward cellular toxicity and inflammation. The RTX adhesin is encoded by other human isolates and is recognized by antibodies present in serum isolated from CF patients colonized by A. xylosoxidans, indicating this virulence factor is produced and deployed in vivo. This study represents the first characterization of A. xylosoxidans replication during infection and identifies a variety of genes that may be linked to virulence and human pathology.IMPORTANCE Patients affected by CF develop chronic bacterial infections characterized by inflammatory exacerbations and tissue damage. Advancements in sequencing technologies have broadened the list of opportunistic pathogens colonizing the CF lung. A. xylosoxidans is increasingly recognized as an opportunistic pathogen in CF, yet our understanding of the bacterium as a contributor to human disease is limited. Genomic studies have identified potential virulence determinants in A. xylosoxidans isolates, but few have been mechanistically studied. Using our optimized in vitro cell model, we identified and characterized a bacterial adhesin that mediates binding and uptake by host macrophages leading to cytotoxicity. A subset of serum samples from CF patients contains antibodies that recognize the RTX adhesion, suggesting, for the first time, that this virulence determinant is produced in vivo. This work furthers our understanding of A. xylosoxidans virulence factors at a mechanistic level.

Molecular Identification of Virulence Genes of Pathogenic Escherichia coli Isolated from Broilers Chicken

Molecular Identification of Virulence Genes of Pathogenic Escherichia coli Isolated from Broilers Chicken

Identification of virulence genes and antimicrobial resistance in Campylobacter spp. from sheep from the state of Pernambuco in Brazil

The objective of this study was to genetically identify virulence and antimicrobial resistance in DNA from Campylobacter spp. from sheep in the state of Pernambuco, Brazil. The presence of virulence genes was investigated from the polymerase chain reaction. The genetic profile of antimicrobial resistance in samples of sheep origin was investigated by sequencing of the 23S rDNA region to identify A2074G and A2075G mutations and gyrA gene fragments to identify C257T and A256G mutations. Forty samples of Campylobacter spp. Of these, 11 were from Campylobacter jejuni, 12 from Campylobacter fetus subsp. fetus and 17 Campylobacter coli from sheep herds. In virulence analysis, 37 samples (92.50%) were positive for the cdtA gene, 30 (75.00%) for cdtB and 28 (70.00%) for cdtC. In the cadF gene research, 38 (95.00%) samples were positive. For the racR, dnaJ and ciaB genes, 32 (80.00%), 19 (47.50%) and 8 (20.00%) positivity were respectively. Only one sample presented the pldA gene and none presented wlaN and virB11. In genotypic analysis of antimicrobial resistance, all samples had the C257T mutation in the gyrA gene, but the A256G mutation was absent. Mutations in 23S rDNA, A2074G and A2075G were also not identified. From the results obtained, we can observe the presence of most virulence genes researched, with high resistance to fluoroquinolones. Thus, studied samples of Campylobacter spp. demonstrated the potential to cause infection and stay in the hosts.

Molecular characterization of virulence (lmb, bca and rib) and macrolid resistance genes (ermB, ermTR and mefA) in clinical isolates of Streptococcus agalactiae

The aim of the study was to molecularly identify virulence and macrolide resistance genes in clinical isolates of Streptococcus agalactiae (GBS), recovered in 2019 from vaginal discharge (n=9) and urine (n=22), from two health facilities in Lima. Identification and antimicrobial susceptibility were determined by the Vitek® 2 automated system, identification was confirmed phenotypically; macrolide resistance was determined by the D-test method. Identification of virulence genes (lmb, bca and rib) and macrolide resistance genes (ermB, ermTR and mefA) was carried out by polymerase chain reaction (PCR). The predominant macrolide resistance phenotype and genotype were cMLSb (12/31) and ermB (11/31); the most frequent virulence gene was lmb (23/31). All were sensitive to penicillin, ampicillin and vancomycin. These findings show the need to implement molecular epidemiology studies that allow adequate knowledge and follow-up of GBS in Peru.

Investigation of Citrus HLB Symptom Variations Associated with “Candidatus Liberibacter asiaticus” Strains Harboring Different Phages in Southern China

Huanglongbing (HLB) is a devastating disease affecting citrus production worldwide. In China, the disease is associated with an unculturable alpha-proteobacterium, “Candidatus Liberibacter asiaticus” (CLas). Phages/prophages of CLas have recently been identified through intensive genomic research. The phage information has facilitated research on CLas biology such as population diversity and virulence gene identification. However, little is known about the roles of CLas phages in HLB symptom development. Such research is challenging due to the unculturable nature of CLas and the lack of laboratory strains that carry a single phage. In this study, CLas strains singly carrying Type 1 phage (Type 1 CLas) and Type 2 phage (Type 2 CLas) were identified and maintained in an experimental screenhouse in southern China. The strains were characterized through next-generation sequencing (NGS). Then, each CLas strain was inoculated into seedlings of three different citrus cultivars/species through graft transmission in a screenhouse in Guangdong, China. Symptom developments were recorded. All CLas-infected cultivars showed HLB symptoms in seven months. In cultivar Nianju (Citrus reticulata), Type 1 CLas caused pronounced yellowing symptoms and severe defoliation, whereas Type 2 CLas caused typical Zn-deficiency-like symptoms. In contrast, symptoms from the two CLas strains’ infections on cultivars Shatianyu (C. maxima), and Eureka lemon (Citrus limon) were more difficult to differentiate. Results from this study provide baseline information for future research to investigate the roles of CLas phages in HLB symptom development.

Isolation, identification and virulence determinants of Streptococcus agalactiae from bovine subclinical mastitis in Egypt

This study aimed to investigate the prevalence of contagious mastitis caused by Streptococcus agalctiae (S. agalactiae) in cattle from households and small-scale dairy farms in Egypt. Molecular characterization of S.agalactiae isolates was described including the genetic determinants of virulence to assess the genetic variation in isolated strains of S. agalactiae. Three hundred and sixty milk samples were collected from 90 apparently healthy dairy cows randomly selected from household and small-scale dairy farms were examined by Somatic Cell Count (SCC) as an indicator for subclinical mastitis. S.agalactiae isolates were bacteriologically and molecularly identified followed by identification of virulence genes using PCR. A total of 172 milk samples (47.77%) were positive with SCC > 200×103/ml. Bacteriological examination of the positive SCC milk samples revealed that 28 (16.28%) of the isolates were S.agalactiae. Molecular examination using PCR confirmed only 22 isolates of S. agalactiae (12.8%). Moreover, we used the pattern of virulence genes to address the genetic variation of S. agalactiae strains isolated from cases of contagious mastitis in cattle in Egypt. Virulence genes hylB, cylE, iagA, and bac were determined in 100%, 68.2%, 13.6% and 100% of isolates respectively. The use of molecular methods for the identification of the causative agent in mastitis confirmed that, in Egypt, Streptococcus agalactiae is considered as one of the predominant infectious agents among contagious mastitis causing pathogens. The pattern of virulence genes presented the genetic diversity of highly virulent S. agalactiae strains isolated from cases of contagious mastitis in cattle in Egypt.

Genome based phylogeny and virulence factor analysis of mastitis causing Escherichia coli isolated from Indian cattle

Mastitis is a highly infectious disease prevalent in dairy cattle and it is majorly caused by Escherichia coli (E. coli). The objective of present study is to investigate the occurrence of virulence genes, antimicrobial susceptibility and comparative analysis of E. coli (IVRI KOL CP4 and CM IVRI KOL-1) isolates from mastitis infected animal. Whole-genome sequencing (WGS) was performed using a PacBio RS II system and de novo assembled using Hierarchical Genome Assembly Process (HGAP3). Bacterial Pan Genome Analysis Pipeline (BPGA) was used for pangenome analysis. A set of 50 E. coli isolates were used for comparative analysis (48 collected from the database and 2 reference sequences). Core genes were further concatenated for phylogenetic analyses. In silico analysis was performed for antibiotic resistance and virulence gene identification. Both of the E. coli isolates carried many resistance genes including, b-lactamase, quinolones, rifampicin, macrolide, aminoglycoside and phenicols resistance. We detected 39 virulence genes in IVRI KOL CP4 and 52 in CM IVRI KOL-1 which include toxins, adhesions, invasins, secretion machineries or iron acquisition system. High prevalence of mastitis strains belongs to phylogroups A, although few isolates were also assigned to phylogenetic groups B1 and B2. In conclusion, the present study reported the presence of genes involved in Adherence, Iron acquisition, secretion system and toxins which shown to be crucial in MPEC pathogenicity. This is the first whole genome analysis of MPEC strains to be carried out in Indian isolate to highlights the spread of resistance and virulence genes in food animals.

Choice of library preparation affects sequence quality, genome assembly, and precise in silico prediction of virulence genes in shiga toxin-producing Escherichia coli.

Whole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. Foodborne pathogens are often sequenced using rapid library preparation chemistries based on transposon technology; however, this method may miss random segments of genomes that can be important for accurate downstream analyses. As new technologies become available, it may become possible to achieve better overall coverage. Here we compare the sequence quality obtained using libraries prepared from the Nextera XT and Nextera DNA Prep (Illumina, San Diego, CA) chemistries for 31 Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains, which had been isolated from flour during a 2016 outbreak. The Nextera DNA Prep gave superior performance metrics including sequence quality, assembly quality, uniformity of genome coverage, and virulence gene identification, among other metrics. Comprehensive detection of virulence genes is essential for making educated assessments of STECs virulence potential. The phylogenetic SNP analysis did not show any differences in the variants detected by either library preparation method which allows isolates prepared from either library method to be analysed together. Our comprehensive comparison of these chemistries should assist researchers wishing to improve their sequencing workflow for STECs and other genomic risk assessments.