Potential Pathogens Research Articles

Comparison of pathogenicity factors of avian pathogenic and extraintestinal pathogenic Escherichia coli isolates originating from broiler chickens

ABSTRACT 1. E. coli is an opportunist pathogen of animals, including food-producing ones and humans. Chickens may be a notable source of pathogenic and antimicrobial resistant E. coli for transmission to humans. 2. This study compared virulence-associated genes (VGs) and antimicrobial resistance (AMR) in avian pathogenic E. coli (APEC) and extraintestinal pathogenic E. coli (ExPEC) isolates from broiler chickens, specifically APEC isolates in liver samples (n = 78) and ExPEC or non-ExPEC isolates in litter samples (n = 34). Virulence was evaluated by PCR for feoB, hlyF, iroN, iss, iutA and ompT genes, while AMR was evaluated by using antimicrobials from seven classes and detecting blaSHV, blaTEM, blaOXA, qnrB, stcM, mrc1, mrc2, sul1 and tetA genes. 3. The APEC isolates were found in 100% of livers, while ExPEC and non-ExPEC isolates were found in 44% and 56% of the litter samples. The predominant VG was feoB (100%), followed by ompT (63%), iutA (60%), iss (58%) and hlyF (43%). Surprisingly, iroN, omp T and iutA had higher prevalences in APEC isolates (85%, 96% and 96%, respectively) than in ExPEC isolates (73%, 87% and 73%, respectively) and non-ExPEC isolates (0% for all). The presence of all VG in 33% of isolates indicated high pathogenicity. 4. The isolates were phenotypically resistant to ampicillin (93%), ceftazidime (72%) and nalidixic acid (82%). All APEC and ExPEC isolates (100%) were multidrug resistant (MDR), while 63% of non-ExPEC isolates were MDR. Genotypic AMR testing revealed that 53% and 52% of all isolates had stcM and tetA, respectively. No isolate was positive for blaSHV, blaOXA, mrc1 or mrc2, which suggested the benefits of colistin for treating carbapenem-resistant enteric pathogens, due to the high resistance detected to meropenem (47%). 5. Given the potential pathogenicity of E. coli isolates, improving biosecurity practices in chicken flocks should be prioritised to eliminate transmission to humans through the food chain.

Development of a conditional plasmid for gene deletion in non-model Fusobacterium nucleatum strains.

Fusobacterium nucleatum is an opportunistic pathogen with four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA), each with distinct disease potentials. Research on fusobacterial pathogenesis has mainly focused on the model strain ATCC 23726 from FNN. However, this narrow focus may overlook significant behaviors of other FNN strains and those from other subspecies, given the genetic and phenotypic diversity within F. nucleatum. While ATCC 23726 is highly transformable, most other Fusobacterium strains exhibit low transformation efficiency, complicating traditional gene deletion methods that rely on non-replicating plasmids. To address this, we developed a conditional plasmid system in which the RepA protein, essential for replication of a pCWU6-based shuttle plasmid, is controlled by an inducible system combining an fdx promoter with a theophylline-responsive riboswitch. This system allows plasmid replication in host cells upon induction and plasmid loss when the inducer is removed, forcing chromosomal integration via homologous recombination in the presence of the antibiotic thiamphenicol. We validated this approach by targeting the galK gene, successfully generating mutants in FNN (ATCC 23726, CTI-2), FNP (ATCC 10953), FNA (21_1A), and the closely related species Fusobacterium periodonticum. Incorporating a sacB counterselection marker in this conditional plasmid enabled the deletion of the radD gene in non-model strains. Interestingly, while radD deletion in 23726, 10953, and 21_1A abolished coaggregation with Actinomyces oris, the CTI-2 mutant retained this ability, suggesting the involvement of other unknown adhesins. This work significantly advances gene deletion in genetically recalcitrant F. nucleatum strains, enhancing our understanding of this pathogen.IMPORTANCEFusobacterium nucleatum is implicated in various human diseases, including periodontal disease, preterm birth, and colorectal cancer, often linked to specific strains and reflecting the species' genetic and phenotypic diversity. Despite this diversity, most genetic research has centered on the model strain ATCC 23726, potentially missing key aspects of other strains' pathogenic potential. This study addresses a critical gap by developing a novel conditional plasmid system that enables gene deletion in genetically recalcitrant strains of F. nucleatum. We successfully deleted genes in the FNN clinical strain CTI-2, the FNA strain 21_1A, and F. periodonticum for the first time. Our findings, particularly the varying behavior of the radD gene production in coaggregation across strains, underscore the complexity of F. nucleatum and the need for broader genetic studies. This work advances our understanding of F. nucleatum virulence at the strain level and provides a valuable tool for future bacterial genetics research.

Evaluation of the recovery effects of antibiotic-resistant lactiplantibacillus plantarum subsp. Plantarum ATCC14917 on the antibiotic-disturbed intestinal microbiota using a mice model.

Supplementing Lactobacillus alongside antibiotic treatment was a curative strategy to modulate gut microbiota and alleviate antibiotic-associated dysbiosis. But the lactobacilli that are used as probiotics are sensitive or have a low level of resistance to antibiotics, so they usually cannot achieve their beneficial effect, since they are killed by the applied antibiotics. This work aimed to develop the highly resistant Lactiplantibacillus plantarum subsp. plantarum ATCC14917 to cephalexin and evaluate its recovery effects of antibiotic-resistant L. plantarum on the antibiotic-disturbed intestinal microbiota using a mice model. After successive growth in LSM broth containing a gradually increasing concentration of cephalexin for 70 days, the minimum inhibitory concentration (MIC) of L. plantarum ATCC14917 to cephalexin significantly increased from 16 μg·mL-1 to 8192 μg·mL-1, but stabilized on 4096 μg·mL-1. After sequencing and sequence analysis, no mutated genes were detected on mobile elements, showing that horizontal transfer of mutated genes could not occur. Compared to the control group (Con), feeding mice with cephalexin (1mg·mL-1; Cep) led to a decrease in alpha diversity. However, concurrently used cephalexin and L. plantarum (Cep+LpR) increased the alpha diversity in both microbial richness and diversity. The Cep+LpR group showed a lowest distance with the Con group than either Cep or Cep+LpS groups, suggesting that resistant L. plantarum treatment was more effective than the original strain for the recovery of intestinal microbiota. Compared to the cephalexin-treated group, concurrent ingestion of cephalexin together with resistant L. plantarum significantly increased the proportion of beneficial bacteria, decreased Firmicutes/Bacteroidetes ratio and abundance of potential pathogens. The use of antibiotic-resistant Lactiplantibacillus plantarum ATCC14917 contributed to a much faster and richer recovery of the gut microbiota disturbed by antibiotic treatment compared to original strain.

Klebsiella pneumoniae employs a type VI secretion system to overcome microbiota-mediated colonization resistance

Microbial species must compete for space and nutrients to persist in the gastrointestinal (GI) tract, and our understanding of the complex pathobiont-microbiota interactions is far from complete. Klebsiella pneumoniae, a problematic, often drug-resistant nosocomial pathogen, can colonize the GI tract asymptomatically, serving as an infection reservoir. To provide insight on how K. pneumoniae interacts with the resident gut microbiome, we conduct a transposon mutagenesis screen using a murine model of GI colonization with an intact microbiota. Among the genes identified were those encoding a type VI secretion system (T6SS), which mediates contact-dependent killing of gram-negative bacteria. From several approaches, we demonstrate that the T6SS is critical for K. pneumoniae gut colonization. Metagenomics and in vitro killing assays reveal that K. pneumoniae reduces Betaproteobacteria species in a T6SS-dependent manner, thus identifying specific species targeted by K. pneumoniae. We further show that T6SS gene expression is controlled by several transcriptional regulators and that expression only occurs in vitro under conditions that mimic the gut environment. By enabling K. pneumoniae to thrive in the gut, the T6SS indirectly contributes to the pathogenic potential of this organism. These observations advance our molecular understanding of how K. pneumoniae successfully colonizes the GI tract.

Metabolic tug-of-war: Microbial metabolism shapes colonization resistance against enteric pathogens.

A widely recognized benefit of gut microbiota is that it provides colonization resistance against enteric pathogens. The gut microbiota and their products can protect the host from invading microbes directly via microbe-pathogen interactions and indirectly by host-microbiota interactions, which regulate immune system function. In contrast, enteric pathogens have evolved mechanisms to utilize microbiota-derived metabolites to overcome colonization resistance and increase their pathogenic potential. This review will focus on recent studies of metabolism-mediated mechanisms of colonization resistance and virulence strategies enteric pathogens use to overcome them, along with how induction of inflammation by pathogenic bacteria changes the landscape of the gut and enables alternative metabolic pathways. We will focus on how intestinal pathogens counteract the protective effects of microbiota-derived metabolites to illustrate the growing appreciation of how metabolic factors may serve as crucial virulence determinants and overcome colonization resistance.

Occurrence of <i>Salmonella</i> and presumptive <i>Bacillus cereus</i> in sesame products from Swiss retail stores

Sesame products such as tahini (tahin) or halva (halwa or helva), originating from Arabic cuisine, are becoming increasingly popular in Switzerland. Pathogens, such as Salmonella, can contaminate sesame products, as evidenced by various product recalls. In this study, the occurrence of Salmonella and Bacillus cereus group members was investigated in 100 sesame products (25 sesame seeds, 16 halva, 19 different sesame pastes, 7 sesame bars, 25 hummus, and 8 other products containing sesame) collected from Swiss retail stores. None of the products were positive for Salmonella, whereas B. cereus group members could be detected with bacterial counts between 1×102 and 9×102 CFU/g in 11 out of 100 (11%) products. The 11 isolates identified by matrix-assisted laser desorption ionization-time of flight were whole-genome sequenced with Illumina technology to confirm the identity of the pathogen, determine its toxin gene profile, and perform panC typing. Most of the isolates harbored genes encoding the enterotoxins Nhe, Hbl, and CytK. The isolates were assigned to diverse B. cereus group members, including one identified as B. cytotoxicus. In addition, one of the isolates matched genetically with the Thurigiensis strain used in biopesticide products. In conclusion, none of the investigated sesame products contained significant levels of Salmonella or B. cereus group members. However, as B. cereus with pathogenic potential was detected in multiple samples, proper storage is crucial to prevent its growth and ensure consumer safety, especially for products with high water activity such as hummus.

Genomic, Evolutionary, and Pathogenic Characterization of a New Polerovirus in Traditional Chinese Medicine Viola philippica.

Viola philippica, a medicinal herbaceous plant documented in the Chinese Pharmacopoeia, is a promising candidate for research into plant-derived pharmaceuticals. However, the study of newly emerging viruses that threaten the cultivation of V. philippica remains limited. In this study, V. philippica plants exhibiting symptoms such as leaf yellowing, mottled leaves, and vein chlorosis were collected and subjected to RNA sequencing to identify potential viral pathogens. A novel polerovirus, named Viola Philippica Polerovirus (VPPV), was identified in V. philippica. VPPV possesses a linear, positive-sense, single-stranded RNA genome consisting of 5535 nucleotides (nt) and encodes seven highly overlapping open reading frames (ORFs). Two potential recombination events were identified within ORF2, ORF3a, and ORF3, providing insights into the genetic diversity and evolution history of this novel polerovirus. An infectious cDNA clone of VPPV was successfully constructed and shown to infect Nicotiana benthamiana. Using a PVX-based heterologous expression system, the VPPV P0 protein was shown to trigger a systemic hypersensitive response (HR)-like reaction in N. benthamiana, indicating that P0 functions as the main pathogenicity determinant. These findings contributed to the detection and understanding of pathogenic mechanisms and control strategies for VPPV in V. philippica.

Whole-exome sequencing identifies novel mutation in intrahepatic cholestasis of pregnancy: A case report and literature review.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disease characterized by pruritus and elevated total bile acid (TBA) levels. The most serious impact of ICP is sudden unexplained intrauterine fetal death, especially when an associated TBA ≥ 100 µmol/L is confirmed.We report a case of a 27-year-old female patient with early-onset severe refractory ICP. Whole-exome sequencing and mutation analyses were performed to obtain genetic data on the patient and her mother. Sanger sequencing was performed to screen the mutation site. Computer-based algorithms were applied to predict the pathogenesis of the identified mutation. Subsequently, we conducted a literature review to characterize the pathological features and perinatal management of severe refractory ICP, especially ICP with genetic susceptibility.A heterozygous mutation in the ABCD3 gene: c.130C > T/p.Pro44Ser was detected in this patient. Through the analysis of pathogenicity prediction software, the mutations were disease-causing. This is the first report to identify the novel p.Pro44Ser mutations of ABCD3 gene in ICP patients.Our report provides new insights into the genetic architecture of ICP involving ABCD3 variants. Early-onset severe refractory ICP is rare and mutations in bile acid metabolism genes might accentuate the phenotype. Emphasized perinatal management and screening for potential pathogenicity sites of variants that drive specific recognition of ICP is necessary.

Effects of Dietary Tea Polyphenols on the Growth, Antioxidant Status, Immune Function, and Intestinal Microbiota of Largemouth Bass (Micropterus salmoides).

This research aimed to explore the impact of tea polyphenol (TP) supplementation on the development, antioxidant properties, immune responses, and gut wellness in largemouth bass (Micropterus salmoides, LMB). Four diets with varying levels of TPs (0.00%, 0.02%, 0.04%, and 0.08%) were devised to feed LMB with an initial weight of 4.3 ± 0.02 g for 56 days, among which the intermittent feeding (IF) group was fed a diet supplemented with TP8 for 7 days, followed by a basal diet for another 7 days, and this was repeated until the end (56th day). The results demonstrated that supplementation with 0.04% or 0.08% TPs in the diet could reduce the crude lipid content and increase the crude protein content of LMB (p < 0.05). The levels of total cholesterol (CHO) and low-density lipoprotein (LDL) in the serum significantly decreased with the addition of 0.08% dietary TPs to the diet (p < 0.05). Dietary TPs can stimulate the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) and enhance the antioxidant capacity of LMB (p < 0.05). The activities of immune enzymes such as acid phosphatase (ACP) were increased to improve the immune response via the addition of TPs (p < 0.05). Supplementation with 0.02% and 0.04% TPs reduced liver fatty infiltration and alleviated hepatocyte damage. Compared with the control diet, dietary TPs significantly increased villus height (VH), villus width (VW), and lipase (LPS) activity in the intestine (p < 0.05), and supplementation with 0.04% TPs significantly increased muscular layer thickness (MT) (p < 0.05). With the increase in dietary TPs, distinct differences were observed in the intestinal microbial composition and the relative abundance of potential pathogens, especially Clostridiaceae, which decreased, along with the enrichment of pathways related to metabolism, including amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Additionally, intermittent feeding could alleviate the adverse effects caused by a high dosage. In conclusion, dietary TPs of LMB could enhance antioxidant capacity and immunity and improve intestinal health, and intermittent feeding could mitigate the adverse effects caused by a high dosage.

Virulent and multidrug-resistant Aeromonas in aquatic environments of Kerala, India: potential risks to fish and humans.

Aeromonas inhabit diverse aquatic habitats and are recognized as both opportunistic and primary pathogens of fish and humans. This study delineates the biochemical and gyrB sequence-based molecular identification of 14 Aeromonas strains isolated from aquatic environments in Kerala, India, identifying them as A. dhakensis (50%), A. hydrophila (28.6%), and A. jandaei (21.4%). These strains exhibit a high prevalence of virulence genes (act, flaA, ser, gcat, lip, and ela) implicated in pathogenesis in both fish and humans. These findings underline the emergence of A. dhakensis, often misidentified as A. hydrophila, as a potential pathogen, highlighting the necessity for comprehensive identification methods. Significantly, all strains demonstrated beta-hemolysis and moderate to strong biofilm formation, enhancing their infectivity potential. Moreover, all isolates exhibited multidrug resistance, with a multiple antimicrobial resistance (MAR) index ranging from 0.39 to 0.56, and a significant presence of class 1 (500-1100bp) and class 2 (250-700bp) integrons, indicating their potential risk to both fish and human populations. Our results underscore the role of aquatic environment as a repository for virulent and multidrug-resistant Aeromonas spp., emphasizing the imperative for prudent antimicrobial usage and regular monitoring of antimicrobial resistance (AMR) in these environments.

Pathogenic potential of ornithogenic &lt;i&gt;Escherichia coli&lt;/i&gt; strains detected in the Earth's polar regions

Introduction. Pathogenic strains of Escherichia coli are an important object of surveillance within the One Health concept in the wild, agriculture and human society. Migratory bird colonies and high latitude avian colonies may be points of active intraspecies and interspecies contact between different animal species, accompanied by the spread of pathogens. At the same time, the phylogeography of E. coli in relation to the presence of natural foci of colibacillosis in polar regions remains virtually unstudied. The aim of this study was to assess the pathogenic potential of E. coli strains from the polar regions of the Earth, based on the analysis of the genomes of these bacteria from typical ornithogenic ecosystems of the Arctic and Antarctic. Materials and methods. The study used collections of E. coli isolated from ornithogenic biological material during expeditions to high latitude areas of the Arctic (archipelagos of Novaya Zemlya, Franz Josef Land, Svalbard) and Antarctic (Haswell Archipelago). 16 cultures associated with avian E. coli (12 polar and 4 temperate strains) were selected for genome-wide sequencing using BGI technology. The annotation of the genomes focused on the identification of genes for pathogenicity factors and antimicrobial resistance, as well as the identification of strains belonging to individual genetic lineages using the cgMLST method. Results. The annotation of the genomes allowed their assignment to different sequence types in the multilocus sequencing typing and genome-wide sequencing typing schemes. The analysis of the geographical distribution of the sequence types of polar E. coli strains determined by the cgMLST method showed their global representation in geographically distant regions of the planet. For example, cgST 133718 was observed in Antarctica (strain 17_1myr) and in the UK, and sequence 11903, to which strain 32-1 from the northernmost point of Novaya Zemlya belonged, was previously identified in the USA. All strains studied were characterized by the presence of extensive virulence. Among the pathogenicity factors identified were haemolysins A, E, F, siderophores, including the yersiniabactin gene cluster, a number of adhesion, colonization and invasion factors, as well as the thermostable enterotoxin EAST-1 and genes that characterize enteroaggregative strains of E. coli (the virulence regulator gene eilA and enteroaggregative protein (air)). One of the Arctic strains (33-1) had determinants of antibiotic resistance, in particular the extended-spectrum beta-lactamase gene TEM-1b and the Tn1721 transposon, including tetracycline resistance genes (tetA-TetR), were detected in its genome. Conclusion. The results of the study indicate the circulation of E. coli strains with strong pathogenic potential in high-latitude Arctic and Antarctic ornithogenic ecosystems. The analysis of genomic data indicates the presence of geographically widespread genetic lineages in these regions, which justifies the importance of monitoring epidemic clones of E. coli, along with monitoring for other pathogens, in bird colonies in high-latitude areas.

Pseudocitrobacter cyperus, a novel bacterial species recovered from Cyperus alternifolius in Egypt

BackgroundStrain Cyp38ST was isolated as an endophyte from the plant Cyperus alternifolius, collected along the banks of the River Nile in 2019. Preliminary analysis tentatively identified Cyp38ST as belonging to the genus Pseudocitrobacter.MethodsThe preliminary identification of Cyp38ST was performed using the VITEK®2 identification system, MALDI-TOF-MS, and 16S rRNA gene sequencing. To confirm its taxonomic classification, the draft genome of Cyp38ST was generated using DNBseq, and the genome-based taxonomic evaluation was conducted by calculating the overall genome-relatedness indices (OGRIs) such as Average Nucleotide Identity (ANI), digital DNA-DNA hybridization (dDDH), and the tetra-nucleotide signatures (Tetra). Additionally, the biochemical features, antimicrobial susceptibility profiles, and fatty acid methyl ester content of Cyp38ST were characterized.ResultsVITEK®2 misidentified Cyp38ST as Citrobacter werkmanii, MALDI-TOF-MS identified it as Pseudocitrobacter faecalis. While the 16S rRNA gene showed more than 99.0% similarity to other Pseudocitrobacter species, the calculated OGRIs were lower than the thresholds recommended for species assignment to all currently known Pseudocitrobacter species. Furthermore, the phylogenomic analysis revealed that Cyp38ST forms a distinct species cluster within the genus Pseudocitrobacter. Cyp38ST was predicted as a potential human pathogen and carried a unique ß-lactamase-coding gene.ConclusionHere we present Cyp38ST (= CCASU-2024-73T) as the type strain of a novel species within the genus Pseudocitrobacter to which we propose the name Pseudocitrobacter cyperus sp. nov. We provide a full description of the novel species and present its genome sequence and annotation. The discovery of this novel species highlights the potential of endophytic bacteria associated with unique plant hosts to harbor previously uncharacterized microbial diversity.

The value of metagenomic next-generation sequencing in the diagnosis of fever of unknown origin

Fever of unknown origin (FUO) caused by infection is a disease state characterized by complex pathogens and remains a diagnostic dilemma. Metagenomic next-generation sequencing (mNGS) technology is a promising diagnostic tool for identifying pathogenic microbes of FUO caused by infection. Little is known about the clinical impact of mNGS in the etiological diagnosis of FUO. This study focuses on the value of mNGS in the etiologic diagnosis of FUO by diagnostic performance, further clarifying the value of mNGS in clinical management. In a single-centre retrospective cohort study, 263 FUO patients who underwent both mNGS and culture at the First Affiliated Hospital of Nanchang University were enrolled from December 2020 to February 2023. The sensitivity and specificity of culture and mNGS were analyzed based on the final clinical diagnosis as the gold standard to assess the diagnostic value of mNGS in FUO cases. Among the 263 patients, 69.96%(184/263) cases were diagnosed as infectious diseases, of which lower respiratory tract infections were the most common, accounting for 53.26%(98/184). 30.04%(79/263) cases had a diagnosis of non-infectious disease. From these cases, mNGS identified 150 true-positive cases, 21 false-positive cases, 58 true-negative cases, and 34 false-negative cases. The sensitivity of mNGS in infection diagnosis was much higher than that of culture [81.52%(150/184) vs. 47.28%(87/184)], but the specificity was the opposite[73.42%(58/79) vs. 84.81%(67/79)]. mNGS had a receiver operating characteristic (ROC) curve of 0.775 for infectious disease, which was significantly higher than that of culture (0.661, P < 0.05). mNGS detection revealed that bacteria were the most commonly identified potential pathogens. The top causative pathogens identified were Acinetobacter baumannii. Of the 263 patients with FUO, clinical management of 48.67% (128/263) patients was positively affected by mNGS, and 51.33% (135/263) patients were not affected by mNGS(P = 0.1074). To sum up, infectious diseases are the principal cause of FUO. mNGS could significantly improve the detected pathogen spectrum of FUO caused by infection. However, the FUO disease spectrum is relatively broad, including a large number of non-infectious diseases. Therefore, Further investigation is warranted into the specific clinical scenarios for which mNGS may offer the greatest clinical diagnostic value.

Focus on the outbreaks of invasive group A streptococcal disease

In recent years, particularly since the outbreaks of scarlet fever and invasive group A streptococcal diseases/infections (iGAS) in several European countries in 2022, iGAS has garnered widespread attention. Recently, Japan experienced an outbreak of a specific type of iGAS, streptococcal toxic shock syndrome (STSS). The outbreak was reported under the label"flesh-eating bacteria,"emphasizing the pathogenic potential of group A streptococcus (GAS). Although GAS is not a newly emerging pathogen and remains susceptible to antibiotics, and guidelines exist for the diagnosis and treatment of various clinical manifestations of iGAS, systematic surveillance and research on iGAS are lacking in China. The high mortality rate associated with the STSS outbreak in Japan highlights the need for continued attention to GAS-related diseases, with a focus on emerging trends in iGAS epidemiology, as well as changes in bacterial virulence and transmission dynamics. There is a pressing need to conduct research based on domestic clinical practice to enhance iGAS diagnosis, treatment, and prevention, ultimately to safeguard public health.

Influence of Saccharomyces cerevisiae CNCM I-1077 on the fecal pH, markers of gut permeability, fecal microbiota, and markers of systemic inflammation in sedentary horses fed a high-starch diet.

Thirty mature Quarter Horse geldings were used in a completely randomized 32-d study to test the hypotheses that supplemental live Saccharomyces cerevisiae CNCM I-1077 improves apparent digestion, stabilizes the fecal pH, reduces gut permeability, maintains microbial communities, and decreases inflammation in horses fed a high-starch diet. Horses were stratified by body weight, age, and body condition score (BCS) to one of two treatments: concentrate formulated with 2g starch • kg BW-1 • meal-1 (CON; n=15) or the same concentrate top-dressed with 25g/d Saccharomyces cerevisiae CNCM I-1077 (SC; n=15; 8×108 CFU). Horses were fed individually in stalls every 12h. Between meals, horses were housed in dry lots with ad libitum access to water and Coastal bermudagrass hay. On d0 and 32, BW and BCS were recorded, and blood was collected before feeding and 2, 8, 16, and 24h postmeal on d32 to analyze serum D-lactate. Fecal samples were collected on d0, 16, and 32 at 8, 16, and 24h post-meal for fecal pH and starch content. Intake and fecal production were recorded over 4-d to measure digestibility on d28-31. Whole blood total bacterial counts and 16S fecal microbiota rRNA sequencing were performed at d0, 16, and 32. Results revealed an increased ∆BW in SC horses compared with CON horses (P=0.03), with no change in BCS (P=0.97). D-lactate tended to be greater in SC horses on d32 at 16 and 24h post-meal compared with CON horses (P=0.10). Concentrations of TNFα and LogCCL2 decreased from d0 to d 32 regardless of dietary supplementation (P≤0.02). Fold change of percent reads from d0 in whole blood bacterial 16S rRNA did not differ between groups. Fecal starch was undetectable, and there were no differences in intake or apparent digestibility. Fecal pH tended (P=0.07) to be lower in CON at 0h on d32 (6.03 ± 0.06) than d16 (6.14 ± 0.06). Additionally, pH tended (P=0.09) to be lower in CON (6.03 ± 0.06) than SC (6.16 ± 0.06) at 0h on d32. Supplementation of Saccharomyces cerevisiae CNCM I-1077 maintained Bacteroidales and reduced acidosis-like bacteria like Streptococcus and potential pathogens like Enterobacteriaceae, Stenotrophomonas, and Rhodococcus at d16 (P<0.05). Further, supplementation increased fibrolytic bacteria at d32, such as Ruminococcus, Fibrobacter, and Succinivibrio (P<0.05). These results indicate Saccharomyces cerevisiae CNCM I-1077 increases BW and promotes a more diverse microbiome when hoses are fed ad libitum hay and a high-starch concentrate.

Virulence profile of carbapenem-resistant Klebsiella pneumoniae strains by an in vivo model of Galleria mellonella.

Klebsiella pneumoniae is a significant healthcare-associated pathogen, notable for its diverse virulence and antibiotic resistance profiles. This study aimed to characterize the genotypic and phenotypic diversity of K. pneumoniae isolates and evaluate their virulence using the Galleria mellonella model. Biomass production, metabolic activity, capsule formation, and siderophore production were assessed in 27 K. pneumoniae isolates from hospital-associated infections. Lethality curves were generated using the G. mellonella model, with survival monitored hourly from 16 to 48 hours. The most common sequence types (ST) identified were the high-risk clones ST307 (N = 10), ST512 (N = 8), ST101 (N = 7), and ST661 (N = 2). These STs were associated with distinct K-locus, including KL102, KL107, KL17, and KL39. Most isolates belonged to the O2afg locus (N = 18), with the K. pneumoniae carbapenemase genotype detected in 96.3% of strains. None of the isolates were classified as hypervirulent. Phenotypically, ST661 exhibited the highest biomass production despite showing similar metabolic activity to other STs. A positive correlation was observed between biomass and siderophore production, while capsule production was inversely correlated with biomass. In the G. mellonella model, ST661 demonstrated the highest virulence, resulting in 100% mortality by 48 hours, compared to survival rates of 21.4% for ST101, 38.0% for ST307, and 31.2% for ST512. These findings underscore the pathogenic potential of ST661 isolates with enhanced biofilm production. The G. mellonella model may serve as an effective in vivo system for evaluating the virulence of emerging K. pneumoniae lineages.IMPORTANCEWe demonstrate that the Galleria mellonella model is a useful tool to analyze the virulence of carbapenem-resistant Klebsiella pneumoniae strains. Our findings highlight the pathogenicity of carbapenem-resistant K pneumoniae isolates, particularly the role of the ST661 that, despite being a rare lineage, harbors the blaVIM gene and is associated with high biofilm production and the highest mortality rates.

The Bacterial and pathogenic landscape of African buffalo (Syncerus caffer) whole blood and serum from Kenya

BackgroundAfrican buffalo (Syncerus caffer) is a significant reservoir host for many zoonotic and parasitic infections in Africa. These include a range of viruses and pathogenic bacteria, such as tick-borne rickettsial organisms. Despite the considerations of mammalian blood as a sterile environment, blood microbiome sequencing could become crucial for agnostic biosurveillance. This study investigated the blood microbiome of clinically healthy wild buffaloes in Kenya to determine its applicability in agnostic testing for bacteria in apparently healthy wild animals.MethodsWhole blood and serum samples were collected from 46 wild African buffalos from Meru National Park (30), Buffalo Springs (6) and Shaba (10) National Reserves in upper eastern Kenya. Total deoxyribonucleic acid (DNA) was extracted from these samples and subjected to amplicon-based sequencing targeting the 16 S rRNA gene. The bacteria operational taxonomic units (OTU) were identified to species levels by mapping the generated V12 and V45 regions of 16 S rRNA gene to the SILVA database. These OTU tables were used to infer the microbial abundance in each sample type and at the individual animal level. The sequences for the corresponding OTUs were also used to generate phylogenetic trees and thus infer evolution for the OTUs of interest.ResultsHere, we demonstrate that buffaloes harbor many bacteria in their blood. We also report a diversity of 16 S rRNA gene sequences for Anaplasma and Mycoplasma from individual animals. By sequencing both whole blood and serum in triplicate for each animal, we provide evidence of the differences in detecting bacteria in both sample types.ConclusionsDiverse bacteria, including some potential pathogens, can be found in the blood of clinically healthy wild African buffalo. Agnostic surveillance for such pathogens can be achieved through blood microbiome sequencing. However, considerations for the question being asked for the blood microbiome in wildlife will impact the choice for using whole blood or serum for sequencing.

Genomic Insight into Vibrio Isolates from Fresh Raw Mussels and Ready-to-Eat Stuffed Mussels.

Consuming raw or undercooked mussels can lead to gastroenteritis and septicemia due to Vibrio contamination. This study analyzed the prevalence, density, species diversity, and molecular traits of Vibrio spp. in 48 fresh raw wild mussels (FRMs) and 48 ready-to-eat stuffed mussels (RTE-SMs) through genome analysis, assessing health risks. The results showed Vibrio prevalence rates of 12.5% in FRMs and 4.2% in RTE-SMs, with V. alginolyticus as the most common species (46.7%). It was determined that the seasonal distribution of Vibrio spp. prevalence in the samples was higher in the summer months. The genome sizes of the Vibrio spp. ranged from approximately 3.9 to 6.1 Mb, with the GC contents varying between 41.9% and 50.4%. A total of 22 virulence factor (VF) classes and up to six antimicrobial resistance (AMR) genes were detected in different Vibrio species. The presence of nine different biosynthetic gene clusters (BGCs), 27 prophage regions, and eight CRISPR/Cas systems in 15 Vibrio strains provides information about their potential pathogenicity, survival strategies, and adaptation to different habitats. Overall, this study provides a comprehensive understanding of the genomic diversity of Vibrio spp. isolated from FRM and RTE-SM samples, shedding light on the prevalence, pathogenicity, and toxicity mechanisms of Vibrio-induced gastroenteritis.

Control of Neonatal Diarrhea in Piglets with Reduced Antibiotic Use by Application of a Complementary Feed-A Randomized Controlled Farm Trial.

The objective of this farm trial was to investigate if the consumption of antibiotics could be reduced when piglets showing early signs of neonatal diarrhea were treated with an oral dose of tannin extract derived from sweet chestnut wood. The farm had a very high incidence of neonatal diarrhea among gilt litters. Gilts were randomized into test or control groups in a 1:1 ratio to compare the consumption of antibiotics used for piglets and piglet mortality during the four-week trial period. Control litters were treated with the oral antibiotic paromomycin, while test litters were treated with the complementary feed O-Nella-Protect. The farm trial included 18 gilt litters comprising 254 piglets. In the control group, 100% of the piglets received antibiotic treatment. In the test group, consumption of antibiotics used against diarrhea was reduced by 84% (p = 0.001) and consumption of antibiotics used for other illnesses was reduced by 45% (p = 0.045). In both test and control groups, six piglets died. Microbiological analysis identified both potential bacterial and viral pathogens. In conclusion, the farm trial indicates that even under the challenge of potentially serious bacterial and viral pathogens, a complimentary feed containing a tannin extract can support piglet health and reduce antibiotic consumption.

Bacterial wilt disease alters the structure and function of fungal communities around plant roots

BackgroundFungal communities around plant roots play crucial roles in maintaining plant health. Nonetheless, the responses of fungal communities to bacterial wilt disease remain poorly understood. Here, the structure and function of fungal communities across four consecutive compartments (bulk soil, rhizosphere, rhizoplane and root endosphere) were investigated under the influence of bacterial wilt disease.ResultsThe results showed that bacterial wilt disease caused different assembly patterns of fungal communities in the bulk soil, rhizosphere, rhizoplane and endosphere. Under the influence of bacterial wilt disease, a decreased fungal diversity was observed in the rhizoplane and endosphere, and completely different kinds of fungal genera were enriched in the four compartments. The complexity and stability of fungal networks were less affected, but the number of key fungal members in networks were significantly reduced in diseased samples. Functional predictions based on FUNGuild suggested that with the pathogen infection, saprotrophic fungi were increased in the bulk soil, but pathotrophic fungi (potential plant and animal pathogens) were increased in the rhizosphere, rhizoplane and endosphere.ConclusionThis work provides a deep insight into the effects of bacterial wilt disease on fungal communities along the soil-root continuum, and is helpful to identify plant-associated beneficial fungi to resist plant disease.Clinical trial numberNot applicable.