Genetic Virulence Research Articles

Genomic Diversity and Virulence Factors of Clostridium perfringens Isolated from Healthy and Necrotic Enteritis-Affected Broiler Chicken Farms in Quebec Province

Avian necrotic enteritis due to the Gram-positive bacterium Clostridium perfringens has re-emerged following the ban on antibiotic growth promoters in many poultry producing countries. The limited number of previous studies has left important gaps in our understanding of the genetic diversity and virulence traits of the pathogen. To address these knowledge gaps, in this study, we sequenced the genomes of 41 Clostridium perfringens isolates recovered from commercial broiler chicken flocks in Quebec, Canada, including isolates from healthy birds and those affected by necrotic enteritis. We sought to understand the pangenome diversity and interrogated the genomes for key virulence factors involved in necrotic enteritis pathogenesis. On average, the genomes had a GC content of 28% and contained 3206 coding sequences. A variable presence of toxins, degradative hydrolytic enzymes, and collagen-binding proteins was also found. Through pangenome analysis, we revealed a total of 10,223 genes, 652 (6.4%) of which formed the core genome. Additionally, we identified 17 different plasmids, 12 antibiotic resistance genes, and nine prophage regions. Overall, our results demonstrated a relatively high genetic diversity among chicken Clostridium perfringens isolates collected from the same geographical location, offering new insights into potential virulence mechanisms and adaptation of the pathogen within poultry populations.

The ‘Oma’s of the Gammas—Cancerogenesis by γ-Herpesviruses

Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), which are the only members of the gamma(γ) herpesviruses, are oncogenic viruses that significantly contribute to the development of various human cancers, such as Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s lymphoma, Kaposi’s sarcoma, and primary effusion lymphoma. Oncogenesis triggered by γ-herpesviruses involves complex interactions between viral genetics, host cellular mechanisms, and immune evasion strategies. At the genetic level, crucial viral oncogenes participate in the disruption of cell signaling, leading to uncontrolled proliferation and inhibition of apoptosis. These viral proteins can modulate several cellular pathways, including the NF-κB and JAK/STAT pathways, which play essential roles in cell survival and inflammation. Epigenetic modifications further contribute to EBV- and KSHV-mediated cancerogenesis. Both EBV and KSHV manipulate host cell DNA methylation, histone modification, and chromatin remodeling, the interplay of which contribute to the elevation of oncogene expression and the silencing of the tumor suppressor genes. Immune factors also play a pivotal role in the development of cancer. The γ-herpesviruses have evolved intricate immune evasion strategies, including the manipulation of the major histocompatibility complex (MHC) and the release of cytokines, allowing infected cells to evade immune detection and destruction. In addition, a compromised immune system, such as in HIV/AIDS patients, significantly increases the risk of cancers associated with EBV and KSHV. This review aims to provide a comprehensive overview of the genetic, epigenetic, and immune mechanisms by which γ-herpesviruses drive cancerogenesis, highlighting key molecular pathways and potential therapeutic targets.

Complete Genome Sequence and In Vitro Probiotic Assessment of Bacillus subtilis DC-11 Isolated from Traditionally Fermented Idli Batter.

In this study, we reported in vitro probiotic assessment and complete genome sequence of Bacillus subtilis DC-11 isolated from traditionally fermented Idli Batter. The strain was evaluated for probiotic properties, biofilm formation, and antimicrobial compound production. The phenotypic safety was determined by accessing the strain's ability to produce enterotoxins, degrade mucin, and antibiotic sensitivity. Whole genome sequencing (WGS) was performed to identify the strain and determine genetic safety by analyzing the presence of plasmids, antibiotic resistance genes, and virulence factors. In the results, B. subtilis DC-11 showed 88.98% viability in gastric juice, and 98.60% viability in intestinal juice. It showed 18.33 ± 0.44% autoaggregation, 32.53 ± 3.11% adhesion to xylene, 0.98 ± 0.05 OD unit's adhesion to mucin (crystal violet equivalence at 550nm), 21.2 ± 2.3% adhesion to Caco-2 cells, and - 22.3 ± 0.65mV zeta potential. The highest co-aggregation was recorded with Escherichia coli (23.62 ± 0.70%). The strain was found negative for enterotoxin production, mucin degradation, and antibiotic resistance to the commonly used therapeutic antibiotics. It formed a good biofilm and capable of producing antimicrobial peptide subtilosin A with a molecular mass of 3400Da. The peptide has inhibited the growth of methicillin-resistant Staphylococcus aureus (18.6 ± 0.58mm). In genetic safety, no plasmids, antibiotic-resistant genes, and virulence factors were detected. Moreover, the strain showed close similarity with B. subtilis ATCC 6051 and proteins involved in probiotic attributes. In conclusion, B. subtilis DC-11 is safe potential probiotic candidate.

Genetic diversity, morphological characteristics and virulence of Stemphylium species causing tomato leaf spot disease in Uruguay

AbstractTomato leaf spot, caused by the ascomycete fungi Stemphylium spp., including S. lycopersici, S. solani and S. vesicarium, is a severe threat to tomato production worldwide. In Uruguay, the disease has become more prevalent in both field and greenhouse production systems in recent years, primarily due to the use of susceptible tomato varieties. This study focuses on the identification, morphological characteristics, genetic diversity and virulence of Stemphylium isolates responsible for leaf spot in Uruguay's major tomato‐producing regions. Phylogenetic analysis using partial sequences of the rDNA internal transcribed spacer (ITS) and glyceraldehyde‐3‐phosphate dehydrogenase (gpd) genes showed that S. lycopersici is the predominant pathogen, with 33 isolates identified, whereas only three isolates were classified as S. vesicarium. Genetic diversity analysis using inter simple sequence repeat (ISSR) markers identified two main clusters of S. lycopersici isolates that correlate with distinct geographical regions. Cultural and morphological characteristics of S. lycopersici varied significantly and pathogenicity assays revealed a broad spectrum of virulence among S. lycopersici isolates, with some isolates causing extensive necrosis and others showing minimal disease lesions. S. lycopersici primarily penetrates plant tissues through stomata and trichomes, and hyphae proliferate both within and on plant tissues, causing chlorosis and necrosis. Infected tissues exhibit alterations in cell wall composition associated with the incorporation of phenolic compounds. This is the first report characterizing various isolates of S. lycopersici and S. vesicarium responsible for tomato leaf spot in Uruguay.

Genomic epidemiology and genetic characteristics of clinical Campylobacter species cocirculating in West Bengal, India, 2019, using whole genome analysis.

Campylobacter species are the most common pathogens responsible for foodborne gastroenteritis worldwide. India is a region with frequent diarrheal infections and a high level of Campylobacter infection incidence, but the detailed genomic information is limited. This study aimed to characterize 112 isolates of Campylobacter from diarrhea patients at two hospitals in Kolkata, West Bengal, by whole genome analysis. The Campylobacter isolates consisted of 90 C. jejuni, 20 C. coli, and 2 C. lari isolates. Multilocus sequence typing analysis revealed that the largest sequence type (ST) populations were ST-2131 in C. jejuni and ST-830 in C. coli and seven novel STs were found in C. jejuni and one in C. coli. Notably, ST-2131, which is rarely seen elsewhere, was positive for a sialylated LOS-related gene (wlaN +neuA + cstIII) associated with Guillain-Barré syndrome. Antibiotic resistance factors predicted from the genome sequence included blaOXA variants (58.9%), tet(O) (54.5%), tet(W) (0.9%), ant(6)-Ia (0.9%), mutation in GyrA (T86I, T86I+D90N, T86I+P104S, T86I+D90N+P104S) (79.5%), and mutation in 23S rRNA (A2075G) (12.5%). In addition to the high drug resistance of Campylobacter in Kolkata, Campylobacter pathogens were circulating that may be associated with Guillain-Barré syndrome. This study indicates the importance of genomic analysis in the surveillance of pathogens, which provides genomic information on genetic diversity, virulence mechanisms, and determinants of antimicrobial resistance.

The Petri dish under the ice: permafrost pathogens and their impact on global healthcare and antibiotic resistance.

A shallow active layer of soil above the permafrost thaws during the summer months which promotes microbial growth and releases previously confined pathogens which result in bacterial epidemics in circumpolar regions. Furthermore, these permafrost sources harbor several antibiotic resistance genes (ARGs) which may disseminate and pose a challenge for pharmacologists worldwide. The authors examined the potential association between climate change-induced permafrost thawing, and the resulting release of antibiotic-resistant pathogens, as well as the potential impact this can have on global healthcare systems in the long run. A cursory abstract screening was done to rule out any articles that did not have to do with viral pathogens caused by melting permafrost. Articles that were not available in English or that our institutions library did not have full-text access were weeded out by a secondary screen. A comprehensive analysis of 13 relevant studies successfully revealed a wide variety of bacterial genera, including Staphylococcus spp., Pseudomonas spp., Acinetobacter spp., and Achromobacter spp., along with a total of 1043 antibiotic resistance genes (ARGs), with most pertaining to aminoglycosides and beta-lactams, offering resistance via diverse mechanisms such as efflux pumps and enzymatic modifications, within the permafrost isolates. Additionally, mobile genetic elements (MGEs) housing antibiotic resistance genes (ARGs) and virulence factor genes (VFGs), including plasmids and transposons, were also discovered. Permafrost thawing is an underrated healthcare challenge warranting the need for further articles to highlight it alongside concerted efforts for effective mitigation.

Comparative genomics and virulence potential of Campylobacter coli strains isolated from different sources over 25 years in Brazil

BackgroundCampylobacter spp. have been reported as a common cause of gastroenteritis in humans in many countries. However, in Brazil there is insufficient data to estimate the impact of Campylobacter in public health. In light of the importance of this foodborne pathogen, the aim of this study was to perform comparative analyses on 80 Brazilian Campylobacter coli genomes isolated from human feces, animals, the environment, and food. Methods include Average Nucleotide Identity (ANI), Gegenees, genomic plasticity, presence of pathogenicity, resistance, and metabolic islands. In addition, virulence analysis in Galleria mellonella were also performed for 18 selected C. coli strains.ResultsThe ANI values confirmed that all strains belonged to the C. coli species. Phylogenetic analyses demonstrated the evolutionary relationships among the studied strains, highlighting the genetic diversity among them. The differences in shared and deleted regions of the studied genomes were demonstrated, with 16 genomic islands identified, including 4 metabolic islands, 4 resistance islands, and 8 pathogenicity islands. We detected genes associated with chemotaxis, exotoxin production, antimicrobial resistance, stress response, defense mechanisms, and intracellular survival among these islands, highlighting the pathogenic potential of these strains. Two strains isolated from human and one from animal showed high virulence, killing 100% of Galleria mellonella larvae. Two strains isolated from the environment and two isolated from food killed 70–90% of the larvae and were classified as virulent. Three strains isolated from animal, two from human, two from the environment and one from food killed 30% to 60% of the larvae and were considered of intermediate virulence. Campylobacter jejuni ATCC 33291, one strain isolated from human and one from food killed 10 to 20% of the larvae and were considered of low virulence. One strain isolated from food did not kill any larvae and was considered avirulent.ConclusionsThe results obtained highlighted the genetic diversity, pathogenic and virulence potential of many of the C. coli strains studied, contributing for a more complete characterization of this important pathogen recognized as a cause of human gastroenteritis.

Genetic diversity and virulence variability of Sclerotinia sclerotiorum in Eastern and Northeastern India

Genetic diversity and virulence variability of Sclerotinia sclerotiorum in Eastern and Northeastern India

Unraveling the genomic landscape of piscine myocarditis virus (PMCV): mutation frequencies, viral diversity and evolutionary dynamics in Atlantic salmon

Abstract Over a decade since its discovery, piscine myocarditis virus (PMCV) remains a significant pathogen in Atlantic salmon aquaculture. Despite its significant impact, the genomic landscape, evolutionary dynamics, and virulence factors of PMCV are poorly understood. This study enhances the existing PMCV sequence dataset by adding 34 genome sequences and 202 new ORF3 sequences from clinical cardiomyopathy syndrome (CMS) cases in Norwegian aquaculture. Phylogenetic analyses, also including sequences from the Faroe Islands and Ireland reveal that PMCV sequences are highly conserved with distinct clustering by country of origin. Still, single CMS outbreaks display multiple PMCV variants, and although some clustering was seen by case origin, occasional grouping of sequences from different cases was also apparent. Temporal data from selected cases indicated increased sequence diversity in the population. We hypothesize that multiple bottlenecks and changing infection dynamics in the host population, with transfer to naïve individuals over time, represent a continuous selection pressure on the virus populations. No clear relation was found between PMCV variants and the severity of heart pathology. However, specific non-synonymous and synonymous mutations that might impact protein function and gene expression efficiency were identified. An additional factor that may impact PMCV replication is the presence of defective viral genomes, a novel finding for viruses of the order Ghabrivirales. This study provides new insights into PMCV genomic characteristics and evolutionary dynamics, highlighting the complex interplay of genetic diversity, virulence markers, and host-pathogen interactions, underscoring the epidemiological complexity of the virus.

A metagenomic catalogue of the ruminant gut archaeome

While the ruminant gut archaeome regulates the gut microbiota and hydrogen balance, it is also a major producer of the greenhouse gas methane. However, ruminant gut archaeome diversity within the gastrointestinal tract (GIT) of ruminant animals worldwide remains largely underexplored. Here, we construct a catalogue of 998 unique archaeal genomes recovered from the GITs of ruminants, utilizing 2270 metagenomic samples across 10 different ruminant species. Most of the archaeal genomes (669/998 = 67.03%) belong to Methanobacteriaceae and Methanomethylophilaceae (198/998 = 19.84%). We recover 47/279 previously undescribed archaeal genomes at the strain level with completeness of >80% and contamination of <5%. We also investigate the archaeal gut biogeography across various ruminants and demonstrate that archaeal compositional similarities vary significantly by breed and gut location. The catalogue contains 42,691 protein clusters, and the clustering and methanogenic pathway analysis reveal strain- and host-specific dependencies among ruminant animals. We also find that archaea potentially carry antibiotic and metal resistance genes, mobile genetic elements, virulence factors, quorum sensors, and complex archaeal viromes. Overall, this catalogue is a substantial repository for ruminant archaeal recourses, providing potential for advancing our understanding of archaeal ecology and discovering strategies to regulate methane production in ruminants.

Characterization of a Highly Virulent Klebsiella michiganensis Strain Isolated from a Preterm Infant with Sepsis.

Klebsiella michiganensis is an opportunistic pathogen that causes an increasing number of serious infections. This study aimed to investigate the etiology of the severe clinical symptoms of sepsis in preterm infants and the characterization of K. michiganensis isolates. Whole-genome sequencing (WGS) was performed on three strains isolated from an infected preterm infant. Additionally, the genomic sequences of 534 K. michiganensis strains were obtained from the NCBI database. To gain deeper insights into these strains, we utilized the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Pathogen Host Interactions (PHI) database annotation tools for comprehensive gene function analyses. Moreover, the multilocus sequence typing (MLST), EasyCGtree, and virulence factor database (VFDB) were employed to determine the sequence types (STs), construct phylogenetic trees, and identify potential virulence factors. Sequence analysis found that the three isolated strains had identical sequence characteristics and did not correspond to any of the known ST types. Virulence factor analysis revealed that the three strains harbored mrkABCDFHIJ, fimABCDEFGHIK, entABCDEFS, fepABCD, and capsule genes. These virulence factors are likely to play crucial roles in enhancing adhesion and metabolic capabilities, resisting phagocytosis (inducing immune cell damage), and ultimately contributing to prolonged bacteremia. The phylogenetic tree and comparative genomics of virulence factors showed the genetic and virulence factor diversity of the currently reported K. michiganensis strains. We identified a novel strain of K. michiganensis that exhibits high virulence and leads to severe septicemia phenotypes in preterm infants. Furthermore, comparative genomic analysis of previously reported K. michiganensis strains revealed the existence of three clades. This comprehensive analysis provides novel insights into the genetic relationships and virulence factor profiles of diverse strains of K. michiganensis. In future, it will be necessary to investigate the concept of the high virulence of K. michiganensis to determine the treatment method.

Biofilm-mediated antibiotic tolerance in Staphylococcus aureus from spinal cord stimulation device-related infections.

Staphylococcus aureus is a predominant cause of infections in individuals with spinal cord stimulation (SCS) devices. Biofilm formation complicates these infections, commonly requiring both surgical and antibiotic treatments. This study explored the biofilm matrix composition and antimicrobial susceptibility of planktonic and biofilm-growing S. aureus isolates from individuals with SCS-related infections. Whole-genome sequencing (WGS) examined genotypes, virulome, resistome, and the pan-genome structure. The study also analyzed biofilm matrix composition, early surface adhesion, hemolytic activity, and antibiotic-susceptibility testing. WGS revealed genetic diversity among isolates. One isolate, though oxacillin susceptible, contained the mecA gene. The median number of virulence factor genes per isolate was 58. All isolates harbored the biofilm-related icaA/D genes. When assessing phenotypic characteristics, all strains demonstrated the ability to form biofilms in vitro. The antimicrobial susceptibility profile indicated that oxacillin, rifampin, and teicoplanin showed the highest efficacy against S. aureus biofilm. Conversely, high biofilm tolerance was observed for vancomycin, trimethoprim/sulfamethoxazole, and levofloxacin. These findings suggest that S. aureus isolates are highly virulent and produce robust biofilms. In cases of suspected biofilm infections caused by S. aureus, vancomycin should not be the primary choice due to its low activity against biofilm. Instead, oxacillin, rifampin, and teicoplanin appear to be more effective options to manage SCS infections.IMPORTANCESCS devices are increasingly used to manage chronic pain, but infections associated with these devices, particularly those caused by Staphylococcus aureus, present significant clinical challenges. These infections are often complicated by biofilm formation, which protects bacteria from immune responses and antibiotic treatments, making them difficult to eradicate. Understanding the genetic diversity, virulence, and biofilm characteristics of S. aureus isolates from SCS infections is critical to improving treatment strategies. Our study highlights the need to reconsider commonly used antibiotics like vancomycin, which shows reduced activity against biofilm-growing cells. Identifying more effective alternatives, such as oxacillin, rifampin, and teicoplanin, provides valuable insight for clinicians when managing biofilm-related S. aureus infections in patients with SCS implants. This research contributes to the growing evidence that biofilm formation is crucial in treating device-related infections, emphasizing the importance of tailoring antimicrobial strategies to the biofilm phenotype.

PIPdb: a comprehensive plasmid sequence resource for tracking the horizontal transfer of pathogenic factors and antimicrobial resistance genes.

Plasmids, as independent genetic elements, carrying resistance or virulence genes and transfer them among different pathogens, posing a significant threat to human health. Under the 'One Health' approach, it is crucial to control the spread of plasmids carrying such genes. To achieve this, a comprehensive characterization of plasmids in pathogens is essential. Here we present the Plasmids in Pathogens Database (PIPdb), a pioneering resource that includes 792964 plasmid segment clusters (PSCs) derived from 1 009571 assembled genomes across 450 pathogenic species from 110 genera. To our knowledge, PIPdb is the first database specifically dedicated to plasmids in pathogenic bacteria, offering detailed multi-dimensional metadata such as collection date, geographical origin, ecosystem, host taxonomy, and habitat. PIPdb also provides extensive functional annotations, including plasmid type, insertion sequences, integron, oriT, relaxase, T4CP, virulence factors genes, heavy metal resistance genesand antibiotic resistance genes. The database features a user-friendly interface that facilitates studies on plasmids across diverse host taxa, habitats, and ecosystems, with a focus on those carrying antimicrobial resistance genes (ARGs). We have integrated online tools for plasmid identification and annotation from assembled genomes. Additionally, PIPdb includes a risk-scoring system for identifying potentially high-risk plasmids. The PIPdb web interface is accessible at https://nmdc.cn/pipdb.

Evaluation of physical and chemical isolation methods to extract and purify Campylobacter jejuni extracellular polymeric substances.

The pathogenic bacterium Campylobacter jejuni is a major food safety concern as it can form biofilms that increase its survival and infective potential. Biofilms consist of microbial cells and extracellular matrix (ECM), which is made of water and extracellular polymeric substances (EPS), which are critical for structural integrity and pathogenicity. The aim of this study was to optimize a protocol for the isolation of C. jejuni ECM. We employed eight physical and chemical isolation methods to extract and purify ECM, followed by different qualitative and quantitative analyses using gel electrophoresis and spectroscopy. This comprehensive approach enabled the evaluation of ECM composition in terms of polysaccharides, proteins, and extracellular DNA. The isolation methods resulted in different yields and purities of the extracted ECM components. Centrifugation in combination with chemical treatments proved to be most effective, isolating higher concentrations of polysaccharides and proteins. Additionally, extraction with ether solution facilitated the recovery of high-molecular-weight extracellular DNA. Overall, we provide a refined methodology for ECM extraction from C. jejuni. As polysaccharides and proteins participate in biofilm stability and microbial communication, and extracellular DNA participates in genetic exchange and virulence, our study contributes towards a better understanding of the persistence of this pathogen in the food industry.

Genetic variation, structural analysis, and virulence implications of BimA and BimC in clinical isolates of Burkholderia pseudomallei in Thailand

Melioidosis is a life-threatening tropical disease caused by an intracellular gram-negative bacterium Burkholderia pseudomallei. B. pseudomallei polymerizes the host cell actin through autotransporters, BimA, and BimC, to facilitate intracellular motility. Two variations of BimA in B. pseudomallei have been reported previously: BimABp and BimA B. mallei-like (BimABm). However, little is known about genetic sequence variations within BimA and BimC, and their potential effect on the virulence of B. pseudomallei. This study analyzed 1,294 genomes from clinical isolates of patients admitted to nine hospitals in northeast Thailand between 2015 and 2018 and performed 3D structural analysis and plaque-forming efficiency assay. The genomic analysis identified 10 BimABp and 5 major BimC types, in the dominant and non-dominant lineages of the B. pseudomallei population structure. Our protein prediction analysis of all BimABp and major BimC variants revealed that their 3D structures were conserved compared to those of B. pseudomallei K96243. Sixteen representative strains of the most distant BimABp types were tested for plaque formation and the development of polar actin tails in A549 epithelial cells. We found that all isolates retained these functions. These findings enhance our understanding of the prevalence of BimABp and BimC variants and their implications for B. pseudomallei virulence.

Heterogeneity and Genomic Plasticity of Acinetobacter baumannii and Acinetobacter nosocomialis Isolates Recovered from Clinical Samples in India.

Acinetobacter baumannii and Acinetobacter nosocomialis are the imperious pathogens in the intensive care units. We aimed to explore the genomic features of these pathogens to understand the factors influencing their plasticity. Using next-generation sequencing, two carbapenem-resistant A. baumannii (AbaBS-3, AbaETR-4) isolates and a pan-susceptible A. nosocomialis (AbaAS-5) isolate were characterised. All genomes exhibited 94% similarity with a degree of heterogeneity. AbaBS-3 and AbaETR-4 harboured antibiotic resistance gene (ARG) repertoire to most antibiotic classes. Carbapenem resistance was due to blaOXA-23 and blaOXA-66 besides the antibiotic efflux pumps. Diverse mobile genetic elements (MGE), insertion sequences (IS), prophages and virulence determinants with a plethora of stress response genes were identified in all three genomes. Class-1 integron in AbaETR-4, encoded genes that confer resistance to aminoglycosides, phenicol, sulfonamides and disinfectants. Substitutions in LpxACD and PmrCAB of AbaETR-4 confirmed the colistin resistance in vitro. Novel mutations in piuA, responsible for transporting cefiderocol, were found in AbaBS-3 and AbaETR-4. Plasmids carrying toxin-antitoxin systems, ARGs and ISs were present in these genomes. All three genomes harboured diverse protein secretion systems, virulence determinants related to immune evasion, adherence, biofilm formation and iron acquisition systems. AbaAS-5 exclusively harboured serine protease pkf, and CpaA substrate of type-II secretion system but lacked the acinetobactin-iron acquisition system. Our work delivers a holistic genome characterization of A. baumannii, coupled with a trailblazing attempt to study A. nosocomialis from India. The presence of ARGs and potential virulence factors interspersed with MGE is a cause for concern, depicting the dynamic adaptability mediated by genetic recombination.

GENETIC VARIATION OF HOUSEKEEPING GENES IN MULTIDRUG RESISTANT PSEUDOMONAS AERUGINOSA

Background: Pseudomonas aeruginosa is a prominent opportunistic pathogen responsible for nosocomial infections, particularly among immunocompromised individuals. Specific Background: Its ability to develop multiple antibiotic resistance poses a significant clinical challenge, highlighting the need for a deeper understanding of its genetic diversity and virulence factors. Knowledge Gap: While previous studies have explored antibiotic resistance mechanisms, there is limited research on the genetic diversity of Pseudomonas aeruginosa isolates in specific geographic regions, such as Kirkuk. Aims: This study aimed to investigate the genetic diversity of Pseudomonas aeruginosa isolates from clinical samples obtained from Kirkuk Civil Hospitals, utilizing Multi-Locus Sequence Typing (MLST) for genetic analysis. Results: Fourteen P. aeruginosa isolates were confirmed through biochemical tests and the VITEK-2 system, with an alarming 85.71% (12/14) exhibiting antibiotic resistance. Molecular analysis revealed the presence of several housekeeping genes, although some genes did not amplify. Notably, two new serotypes (PS3:id:9797 and PS4:id:9796) were identified and added to the MLST database, along with three new genes registered in NCBI. Phylogenetic analysis indicated a divergent cluster among three isolates. Novelty: This research contributes new insights into the genetic diversity of Pseudomonas aeruginosa, identifying novel serotypes and genes, which are critical for understanding its epidemiology and resistance mechanisms. Implications: The findings underscore the importance of ongoing surveillance of Pseudomonas aeruginosa in clinical settings to inform treatment strategies and public health policies aimed at managing antibiotic resistance and improving patient outcomes.

Prevalence, Virulence Genes, Drug Resistance and Genetic Evolution of Trueperella pyogenes in Small Ruminants in Western China.

Trueperella pyogenes is a significant opportunistic pathogen that causes substantial economic losses in animal agriculture due to its ability to infect various animal tissues and organs. Limited research has been conducted on the prevalence and biological characteristics of T. pyogenes isolated from sheep and goats. This study aimed to isolate T. pyogenes from clinical samples of sheep and goats in western China, examining genetic evolutionary relationships, antibiotic resistance, and virulence genes. Between 2021 and 2023, standard bacteriological methods were used to isolate and identify T. pyogenes from 316 samples (209 from goats and 107 from sheep) collected from 39 farms. Susceptibility to 14 antibiotics was tested using broth microdilution per CLSI guidelines, and PCR detected eight virulence genes. Whole-genome sequencing analyzed genetic relationships and gene carriage status in 39 isolates. The results indicated that 86 strains of T. pyogenes were isolated from 316 samples, yielding an isolation rate of 27.2% (goats n = 47, 22.5%; sheep n = 39, 36.4%). The virulence genes plo, cbpA, nanH, nanP, fimA, fimC, and fimE were present in 100%, 66.7%, 64.1%, 71.8%, 69.2%, 59.0%, and 82.1% of isolates, respectively, with none carrying the fimG gene. The dominant virulence genotype was plo/nanH/nanP/fimA/fimC/fimE. The isolates exhibited resistance to erythromycin (44.2%, 38/86), gentamicin (38.4%, 33/86), sulfamethoxazole/trimethoprim (37.2%, 32/86), tetracycline (32.6%, 28/86), and streptomycin (32.6%, 28/86), and low resistance to chloramphenicol (14.0%, 12/86), ciprofloxacin (7.0%, 6/86), penicillin (5.8%, 5/86), and clindamycin (4.7%, 4/86). All isolates were susceptible to cefotaxime, vancomycin, and linezolid. Among the 86 isolates, 37 (43.0%) displayed multidrug resistance (MDR) characteristics. The whole genome sequencing of 39 isolates identified eight types of resistance genes, including ant(2″)-Ia, ant(3″)-Ia, cmlA1, cmx, erm(X), lnu(A), sul1, and tet(W). Except for tet(W), erm(X), and sul1, the other resistance genes were reported for the first time in T. pyogenes isolated in China. The drug susceptibility test results and resistance gene detection for the isolated strains were consistent for tetracycline, erythromycin, gentamicin, and sulfisoxazole. Similar allelic profiles and genetic evolutionary relationships were found among isolates from different farms. This study highlights the antibiotic resistance status and virulence gene-carrying rate of Trueperella pyogenes, providing a basis for clinical medication.

Clinical impact of major pathogenic genotypes of Pseudomonas aeruginosa associated with refractory chronic suppurative otitis media.

Chronic suppurative otitis media (CSOM) is characterized by persistent inflammation of the mucous membrane of the middle ear and mastoid. One of the primary causative agents of CSOM is P. aeruginosa, known for its production of virulent toxins and enzymes. Some cases of CSOM, improvement may not occur despite treatment lasting three weeks, leading to what is termed refractory CSOM. This research aims to characterize the P. aeruginosa strains isolated from patients with refractory CSOM in Gyeongsangnam-do, South Korea, providing insights into their pathogenic profiles. We conducted a retrospective analysis of P. aeruginosa isolates from the otorrhea of patients diagnosed with CSOM at a tertiary hospital in Gyeongsangnam-do, over a period from January 2005 to August 2022. The strains were examined using multilocus sequence typing (MLST) and toxin gene assay to assess genetic diversity and virulence. 39 samples were obtained from 13 cases of refractory CSOM and 15 cases of non-refractory CSOM. The findings unveiled that the P. aeruginosa cultured from patients with refractory CSOM belonged to the P. aeruginosa sequence type 235 (ST235) strain, which harbors the exoU gene as a major virulence factor. The detection of ST235 in refractory CSOM signifies a challenging clinical scenario. Given the genotype's strong virulence and antibiotic resistance, identifying ST235 through MLST can guide effective management approaches, including potential surgical intervention. This study underscores the necessity of broader epidemiological investigations to understand ST235 behavior and advocates for patient education to mitigate the impacts of this formidable pathogen in CSOM.

Molecular Detection of Beta-Lactamase Genes (KPC and CTX-M) of Pseudomonas aeruginosa in Diabetic Foot Ulcers Patients

Background: diabetic foot ulcers (DFUs ) are an inflammatory disease caused by a bacterial infection such as Pseudomonas aeruginosa (P. aeruginosa), provided with genetic virulence factors. This study aims to detect KPC and CTX-M genes of P. aeruginosa isolated from chronic DFU patients. Methods: One hundred twenty DFU patients were recruited from Al-Sader Medical City and Al-Hakim General Hospital in Najaf province from September to January 2021 to collect ear swaps. The Primary identification depends on Gram stain and biochemical tests. We also used the Vitek 2 system to make another identification and examine resistance to antibiotics. A polymerase chain reaction (PCR) was used to amplify the required genes. Furthermore, gel electrophoresis was performed to show the amplified genes. Results: The results have revealed that 100 samples (83.3%) give a positive outcome, while 20 samples (16.6%)are negative for culture bacteria. Only 17(17 %) of the 100 clinical specimens showed positive tests for P. aeruginosa. However, the gene, CTX-M gene is found in 58.8% of P. aeruginosa isolates, while the KPC gene is not found in the isolates. Conclusion: Our findings suggest that P. aeruginosa is one of the most common causes of DFU infection in Iraqi patients and The levels of MDR of P. aeruginosa isolates in Najaf hospitals were undoubtedly high. Most bacterial isolates show the presence of the CTX-M gene. The presence of these genes is one of the virulence factors and probably partly explains the resistance to antibiotics.