16S rRNA Gene Amplification Research Articles

First Report of Robbsia andropogonis Causing Bacterial Blight of Danae racemosa (Poet’s Laurel) in a Virginia Ornamental Nursery

Danae racemosa (L.) Moench (Asparagaceae, Monocots) is a shade-tolerant, evergreen shrub native to Persia. On 02 July 2024, at an ornamental nursery in Hanover County, Virginia, 300 plants in 7.6 L containers were observed with blotchy, rust-colored, water-soaked lesions on the cladophylls and stems. Heavy bacterial streaming was observed from the lesions under the microscope, and sterilized tissue was cultured on King's B medium at 22 ± 2°C. After 72 hours, cream-colored colonies developed that were oxidase and gram negative and did not produce fluorescent pigments on King B medium. PCR amplification and sequencing of the partial 16S rRNA, gyrB, and rpoD genes from strain GS24-DSB16 were deposited into the NCBI Genbank database (PQ181184, PQ182786, PQ182787). Based on the morphological and molecular characteristics, the causal agent was identified as Robbsia andropogonis (Smith) Lopes-Santos et al. Koch’s postulates were fulfilled by wounding twenty cladophylls from each plant with a sterile needle, and 10 μl of an aqueous bacterial suspension was pipetted on each wound. R. andropogonis has a wide global distribution from a host range spanning >15 plant families, including maize, sorghum, sugarcane, citrus, coffee, and carnation. To the authors knowledge, this is the first report of the bacteria causing blight on D. racemosa, and the first host in Asparagaceae.

12468 Loss Of Enterococcus And Faecalibacterium Associate With Polycystic Ovary Syndrome And Hirsutism In A Sample Of Hispanic Patients

Abstract Disclosure: L.A. Gonzalez-Rodriguez: Research Investigator; Self; Eli Lilly & Company. C.M. Casas Loyola: None. C.I. Martinez-Hernandez: None. L. El Musa Penna: None. J. Romaguera: None. F. Godoy-Vitorino: None. Polycystic Ovary Syndrome (PCOS) is a common endocrine disorder affecting women of reproductive age. It is characterized by symptoms such as irregular menstrual periods, excess androgen levels and polycystic ovaries. Women with PCOS often display insulin resistance independent of fat and have been associated with an increased risk of diabetes mellitus, cardiovascular disease, and mood disorders. The gut microbiota, the community of microbes living in the human gastrointestinal tract, plays a crucial role in human health and has been implicated in various diseases, including metabolic and inflammatory conditions. We aimed to characterize the gut microbiota in a sample of Hispanic women living in Puerto Rico (n=50). Of these 38 had confirmed PCOS as per Rotterdam criteria and 12 were controls. A stool sample was collected for genomic DNA extractions followed by amplification and sequencing of 16S rRNA genes (V4 region) with Illumina MiSEQ. Data analyses were performed with standard pipelines with variables sample group (PCOS vs Controls), insulin resistance assessed using HOMA-IR and hirsutism status according to modified-Ferriman Gallwey (mFG) score and controlled for age and other clinical variables. Despite there were no changes in alpha or beta diversity among the microbiota of these groups, composition was significantly different. Women with PCOS had significantly lower levels of Enterococcus species as well as Hungatella, taxa that are part of a physiologically normal gut flora. Enterococcus species contribute to the balance of the microbial community and play roles in the metabolism and immune function and are very resilient. Hungatella species are a known source of catabolic enzymes that degrade glycosaminoglycans which are important in tissue repair. These dominant taxa in controls was substituted by other taxa in PCOS. Women with prominent hirsutism had significantly lower levels of Faecalibacterium while those with insulin resistance had increased levels of Clostridium. We found that higher levels Clostridium, and lower levels of protective anti-inflammatory taxa such as Faecalibacterium or Enterococci, which are important for gut health and metabolic regulation, are linked to this metabolic disturbance. Even though this is a preliminary report, it opens potential therapeutic strategies, such as the use probiotics, and dietary modifications to modulate the gut microbiome and improve PCOS outcomes. Presentation: 6/1/2024

12468 Loss Of Enterococcus And Faecalibacterium Associate With Polycystic Ovary Syndrome And Hirsutism In A Sample Of Hispanic Patients

Abstract Disclosure: L.A. Gonzalez-Rodriguez: Research Investigator; Self; Eli Lilly & Company. C.M. Casas Loyola: None. C.I. Martinez-Hernandez: None. L. El Musa Penna: None. J. Romaguera: None. F. Godoy-Vitorino: None. Polycystic Ovary Syndrome (PCOS) is a common endocrine disorder affecting women of reproductive age. It is characterized by symptoms such as irregular menstrual periods, excess androgen levels and polycystic ovaries. Women with PCOS often display insulin resistance independent of fat and have been associated with an increased risk of diabetes mellitus, cardiovascular disease, and mood disorders. The gut microbiota, the community of microbes living in the human gastrointestinal tract, plays a crucial role in human health and has been implicated in various diseases, including metabolic and inflammatory conditions. We aimed to characterize the gut microbiota in a sample of Hispanic women living in Puerto Rico (n=50). Of these 38 had confirmed PCOS as per Rotterdam criteria and 12 were controls. A stool sample was collected for genomic DNA extractions followed by amplification and sequencing of 16S rRNA genes (V4 region) with Illumina MiSEQ. Data analyses were performed with standard pipelines with variables sample group (PCOS vs Controls), insulin resistance assessed using HOMA-IR and hirsutism status according to modified-Ferriman Gallwey (mFG) score and controlled for age and other clinical variables. Despite there were no changes in alpha or beta diversity among the microbiota of these groups, composition was significantly different. Women with PCOS had significantly lower levels of Enterococcus species as well as Hungatella, taxa that are part of a physiologically normal gut flora. Enterococcus species contribute to the balance of the microbial community and play roles in the metabolism and immune function and are very resilient. Hungatella species are a known source of catabolic enzymes that degrade glycosaminoglycans which are important in tissue repair. These dominant taxa in controls was substituted by other taxa in PCOS. Women with prominent hirsutism had significantly lower levels of Faecalibacterium while those with insulin resistance had increased levels of Clostridium. We found that higher levels Clostridium, and lower levels of protective anti-inflammatory taxa such as Faecalibacterium or Enterococci, which are important for gut health and metabolic regulation, are linked to this metabolic disturbance. Even though this is a preliminary report, it opens potential therapeutic strategies, such as the use probiotics, and dietary modifications to modulate the gut microbiome and improve PCOS outcomes. Presentation: 6/1/2024

Genotypes and Phylogenetic Analysis of Helicobacter pylori Clinical Bacterial Isolates

Helicobacter pylori is a human pathogen bacterium associated with gastritis, peptic ulcer, and gastric cancer. It can be identified through the 16S rRNA gene and characterized through cagA and vacA virulence genes. Clinical cultures of H. pylori were isolated and identified from human stomach biopsies. The isolates were characterized according to their colonial and microscopic morphology, and molecular genotyping was conducted to determine the bacterial virulence. A phylogenetic analysis of the 16S rRNA gene sequencing was performed. In addition, multilocus sequence typing analysis was performed to determine the phylogeographic nature of the isolated strains. Three bacterial isolates were selected from 22 gastric biopsies, identified as H. pylori through colonial morphology, Gram staining, urease, catalase, and oxidase tests and identification of the ureC gene through end-point PCR. Amplification of 16S rRNA, urea, and tonB genes was performed, as well. Differences between the cagA and vacA genotypes were determined among the isolates. The phylogenetic analysis confirmed the identity of the three isolates as the specie Helicobacter pylori. Different genotypes were obtained for each H. pylori strain, and all the clinical isolates showed the vacA s2/m2 genotype, indicating an absence of the VacA cytotoxin. Only HCGDL-MR01 is a cagA gene carrier with a greater risk to develop a serious disease, such as stomach cancer and peptic ulcer. The multilocus sequence typing placed all the strains within the hpEurope population structure.

Detection and characterization of vector-borne parasites and Wolbachia endosymbionts in greater one-horned rhinoceros (Rhinoceros unicornis) in Nepal

Vector-borne parasite infections affect both domestic and wild animals. They are often asymptomatic but can result in fatal outcomes under natural and human-induced stressors. Given the limited availability of molecular data on vector-borne parasites in Rhinoceros unicornis (greater one-horned rhinoceros), this study employed molecular tools to detect and characterize the vector-borne parasites in rescued rhinoceros in Chitwan National Park, Nepal. Whole blood samples were collected from thirty-six R. unicornis during rescue and treatment operations. Piroplasmida infections were first screened using nested polymerase chain reaction (PCR) targeting 18S ribosomal RNA gene. Wolbachia was detected by amplifying 16S rRNA gene, while filarial nematodes were detected through amplification of 28S rRNA, COI, myoHC and hsp70 genes. Our results confirmed the presence of Theileria bicornis with a prevalence of 75% (27/36) having two previously unreported haplotypes (H8 and H9). Wolbachia endosymbionts were detected in 25% (9/36) of tested samples and belonged to either supergroup C or F. Filarial nematodes of the genera Mansonella and Onchocerca were also detected. There were no significant association between T. bicornis infections and the age, sex, or location from which the animals were rescued. The high prevalence of Theileria with novel haplotypes along with filarial parasites has important ecological and conservational implications and highlights the need to implement parasite surveillance programs for wildlife in Nepal. Further studies monitoring vector-borne pathogens and interspecies transmission among wild animals, livestock and human are required.

First report of occurrence of Babesia gibsoni in captive Indian wolves

Blood samples from fifteen captive Indian wolves (Canis lupus pallipes) maintained at Arignar Anna Zoological Park, Vandalur, Chennai were screened for the presence of Babesia spp., Ehrlichia canis and Trypnosoma evansi DNA by PCR. Out of 15 wolf samples, 3 samples were found positive for Babesia spp. The amplified 18S rRNA gene fragments from 3 wolves were sequenced and confirmed as Babesia gibsoni. A maximum likelihood tree was constructed using the three sequences along with other Babesia spp. sequences derived from GenBank adopting HKY nucleotide substitution model based on the Bayesian Information Criterion. The phylogenetic analysis confirmed that the three sequences were of Babesia gibsoni and highly divergent from Babesia canis, B. vogeli and B. vulpes. This might be a possible spill over event of B. gibsoni from community dogs through blood feeding dog ticks. This is the first report and molecular confirmation of B. gibsoni infection in captive Indian wolves.

Host genetics drives differences in cecal microbiota composition and immune traits of laying hens raised in the same environment

Vaccination is one of the most effective strategies for preventing infectious diseases but individual vaccine responses are highly heterogeneous. Host genetics and gut microbiota composition are 2 likely drivers of this heterogeneity. We studied 94 animals belonging to 4 lines of laying hens: a White Leghorn experimental line genetically selected for a high antibody response against the Newcastle Disease Virus (NDV) vaccine (ND3) and its unselected control line (CTR), and 2 commercial lines (White Leghorn [LEG] and Rhode Island Red [RIR]). Animals were reared in the same conditions from hatching to 42 d of age, and animals from different genetic lines were mixed. Animals were vaccinated at 22 d of age and their humoral vaccine response against NDV was assessed by hemagglutination inhibition assay and ELISA from blood samples collected at 15, 19, and 21 d after vaccination. The immune parameters studied were the 3 immunoglobulins subtypes A, M, and Y and the blood cell composition was assessed by flow cytometry. The composition of the cecal microbiota was assessed at the end of the experiment by analyzing amplified 16S rRNA gene sequences to obtain amplicon sequence variants (ASV). The 4 lines showed significantly different levels of NDV vaccine response at the 3 measured points, with, logically, a higher response of the genetically selected ND3 line, and intermediate and low responses for the unselected CTR control line and for the 2 commercial lines, respectively. The ND3 line displayed also a higher proportion of immunoglobulins (IgA, IgM, and IgY). The RIR line showed the most different blood cell composition. The 4 lines showed significantly different microbiota characteristics: composition, abundances at all taxonomic levels, and correlations between genera and vaccine response. The tested genetic lines differ for immune parameters and gut microbiota composition and functions. These phenotypic differences can be attributed to genetic differences between lines. Causal relationships between both types of parameters are discussed and will be investigated in further studies.

Isolation and characterization of PGPR obtained from different arsenic-contaminated soil samples and their effect on photosynthetic characters of maize grown under arsenic stress.

Contamination of the environment due to speedup of anthropogenic activities has become a serious threat to modern humanity. Among the contaminants, the new emerging concern is the heavy metal (HM) contamination in the environment. Because the persistence and harmfulness of heavy metals affect the ecosystem and the health of plants, animals, and humans, they are the most toxic substances in the environment. Among them, Arsenic (As) emerged as major environmental constraint leading to enormous negative effects on the plant, animal, and human health. Even in minute quantity, As is known to cause various critical diseases in humans and toxicity in plants. Research was performed to observe the capability of plant growth-promoting strains of bacteria in enhancing Zea mays (L.) growth in arsenic polluted soil. Total 30 bacterial strains were isolated from the polluted soils, screened for plant growth promotion potential and arsenic tolerance. Eighteen isolates showed resistance to different levels of sodium arsenate (ranging from 0 to 50 mM) in agar plate using LB media. Of 18 isolates, 83.3% produced IAA, methyl red, and hydrogen cyanide; 55.5% exhibited catalase activity; 61.1% showed siderophore production; 88.8% showed phosphate solubilization; and 44.4% showed oxidase, Voges proskauer activity, and KOH solubility. The most efficient isolates SR3, SD5, and MD3 with significant arsenic tolerance and plant growth-promoting (PGP) activity were examined via sequencing of amplified 16S rRNA gene. Isolates of bacteria, i.e., SR3, SD5, and MD3, showing multiple PGP-traits were identified as Bacillus pumilus (NCBI accession number: OR459628), Paenibacillus faecalis (NCBI accession number: OR461560), and Pseudochrobactrum asaccharolyticum (NCBI accession number: OR458922), respectively. Maize seeds treated with these PGPR strains were grown in pots contaminated with 50 ppm and 100 ppm sodium arsenate. Compared to untreated arsenic stressed plants, bacterial inoculation P. asaccharolyticum (MD3) resulted 20.54%, 18.55%, 33.45%, 45.08%, and 48.55% improvement of photosynthetic pigments (carotenoid content, chlorophyll content, stomatal conductance (gs), substomatal CO2, and photosynthetic rate), respectively. Principal component analysis explained that first two components were more than 96% of the variability for each tested parameter. The results indicate that in comparison to other isolates, P. asaccharolyticum isolate can be used as efficient agent for improving maize growth under arsenic polluted soil.

Molecular and next-generation sequencing analysis of tick-borne pathogens of Rhipicephalus ticks (Acari: Ixodidae) in cattle and dogs

Ticks are small and adaptable arachnid ectoparasites and global carriers of various pathogens that threaten both human and animal health. They are present in many parts of China. A total of 858 ticks were collected from various regions and hosts, then subjected to species identification based on morphological and molecular characteristics, as described in the authors’ previous study. Eighty-three individual tick samples were selected for screening pathogens based on metagenomic next-generation sequencing (mNGS) and polymerase chain reaction (PCR) assays. The genomic DNA of tick species was extracted, and amplification of the bacterial 16S rRNA gene was carried out from DNA of individual ticks using V3-V4 hypervariable regions, before subjecting to metagenomic analysis. Each tick underwent specific PCR tests for identifying the bacterial species present, including Anaplasma, Ehrlichia, Coxiella, and Rickettsia, and also protozoans such as Babesia, Theileria, and Hepatozoon. Illumina NovaSeq sequencing results revealed that the dominant phylum and family in Rhipicephalus spp. were Bacteroidota and Muribaculaceae, respectively. Alpha diversity patterns varied depending on tick sex (R. linnaei only), species and location, but not on host. Furthermore, bacterial pathogens, including A. marginale (58 %, 29/50), A. platys (6 %, 3/50), E. minasensis (2 %, 1/50), Ehrlichia sp. (10 %, 5/50), T. sinensis (24 %, 12/50), T. orientalis (54 %, 27/50) and Coxiella-like bacteria (CLB) (80 %, 40/50) were detected in R. microplus, while E. canis (33.33 %, 10/30), H. canis (20 %, 6/30) and CLB (100 %, 30/30) were detected in R. linnaei. Also, Anaplasma sp. (33.33 %, 1/3), A. marginale (33.33 %, 1/3), R. felis (33.33 %, 1/3) and CLB (100 %, 3/3) were detected in R. haemaphysaloides. Dual and triple co-infections involving pathogens or CLB were detected in 84.00 % of R. microplus, 66.66 % of R. haemaphysaloides, and 33.00 % of R. linnaei. The report on microbial communities and pathogens, which found from Rhipicephalus spp. in Hainan Island, is an important step towards a better understanding of tick-borne disease transmission. This is the first report in the area on the presence of Anaplasma sp., A. marginale, R. felis and Coxiella, in R. haemaphysaloides.

Characterization and in-vitro plant-based control of hindgut bacteria isolated from Odontotermes obesus Rambur (Termitidae) and Heterotermes indicola Wasmann (Rhinotermitidae).

Termites cause a serious menace to wooden structures all over the world. They rely mostly on entozoic fauna residing in their hindgut for the digestion of cellulosic and hemicellulosic materials. One of the ways to control termites is through their gut symbionts. The present study was designed to characterize the hindgut bacteria isolated from Odontotermes obesus and Heterotermes indicola. Furthermore, the growth inhibitory effect of eight tropical plant extracts was investigated to find out potential control agents for these bacterial isolates. The characterization of bacteria was carried out based on their morphology, Gram staining, biochemical and amplification of 16SrRNA gene. Amplified products were sequenced to confirm their relationship with bacterial isolates from termites of other regions. The growth inhibitory effect of ethanolic leaf extracts of eight plants was evaluated in an invitro agar well diffusion method. Qualitative and quantitative phytochemical analysis of the most effective plant was carried out to learn about bioactive agents. The results confirmed the presence of five bacteria from each termite species. The Bacillus cereus, Escherichia coli, and Lysinibacillus fusiformis were common to both termites whereas Lysinibacillus xylanilyticus and Lysinibacillus macrolides were found in O. obesus only and H. indicola harbor Bacillus subtilis and Shigella sonnei in addition to common three ones. Among the plant extracts of Carica papaya, Eucalyptus camaldulensis, Osmium basilicum, Grevillea robusta, Eucalyptus globulus, Pongamia pinnata, Mentha longifolia, and Melia azedarach, the G. robusta > E. camaldulensis > O. basilicum were found to have growth inhibitory effects with increasing concentrations from 100 to 2000 µg/mL. The biodiversity of the bacterial fauna is important for the biological control of termites. Leaf extracts of these medicinal plants can be used to control termite infestation in an environment-friendly manner to save huge economic loss.

584. Microbiological Outcomes of Culture-Negative Blood Specimens Using 16s rRNA Broad-Range PCR Sequencing: a Retrospective Study in a Canadian Province from 2018 to 2022

Abstract Background Broad-range bacterial PCR sequencing (BRBPS) has emerged as a novel tool to detect fastidious organisms. While its utility has been characterized in different specimen types, its role in culture-negative blood specimens remains poorly understood. Methods We reviewed all clinical specimens sent for culture-negative blood BRBPS (blood, serum and blood culture bottles) to the Laboratoire de santé publique du Québec, the reference laboratory for a large Canadian province, from May 2018 to November 2022. Sanger sequencing of the amplified 16s rRNA gene was performed in all PCR-positive specimens. Data were extracted from the laboratory information system, and the analysis was restricted to the first specimen per patient. Microbiological outcomes were categorized as interpretable sequence, uninterpretable sequence, or negative PCR result. Interpretable sequences were identified and then classified based on the National Healthcare Safety Network database (NHSN; Table 1) and microbiological characteristics.Table 1.Definitions of National Health Safety Network (NHSN) Classification and Examples Results A total of 1199 blood specimens were analyzed using BRBPS from 852 unique patients. Of these, there was no PCR amplification in 152, amplification with uninterpretable sequences in 445 and an interpretable sequence in 255 specimens (Figure 1). Blood specimens received at room temperature, in blood culture bottles, or with positive gram stain were more likely to yield interpretable sequences (Table 2). We identified 174 patients with BRBPS results suggestive of organisms associated with mucosal barrier injury (n=89) or possible pathogens (n=85), summarized in Figure 2. In contrast, 75 patients had results suggestive of contamination from common commensal organisms (n=44) or taxa not in the NSHN database (n=31).Figure 1.Flowchart of the analysis from primary specimens to those specimens with successful amplification of 16s rRNA Classification of interpretable sequences using the National Healthcare Safety Network database classification of microorganisms.Table 2.Characteristics of BRBPS of the 16s rRNA on Culture-Negative Blood SpecimensFigure 2.Tree Map of Mucosal Barrier Injury Organisms and Possible Pathogens by Genera Mucosal barrier injury (A) and possible pathogens (B) are categorized based on the National Healthcare Safety Network database. Microorganisms are colour classified based on microbiological characteristics. Conclusion Our findings demonstrate the potential utility of BRBPS in blood specimens from culture-negative patients, particularly infectious syndromes caused by fastidious gram-negative bacteria associated with animal or arthropod exposures or anaerobic bacteria. However, the frequent recovery of commensal and environmental organisms argues for careful and judicious use. Additional technical optimization is likely required to improve diagnostic yield, particularly with mixed sequences. Disclosures Matthew Cheng, MD, Amplyx Pharmaceuticals: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Honoraria|Cidara Therapeutics: Grant/Research Support|GEn1E lifesciences: Advisor/Consultant|GEn1E lifesciences: Stocks/Bonds|Kanvas Biosciences, Inc.: Board Member|Kanvas Biosciences, Inc.: Pending patents|Kanvas Biosciences, Inc.: Ownership Interest|Merck: Honoraria|nomic bio: Advisor/Consultant|nomic bio: Stocks/Bonds|Pfizer: Honoraria|Scynexis Inc.: Grant/Research Support|Takeda: Advisor/Consultant|Takeda: Honoraria

Is Brucella excreted in cattle faeces? – Evidence from Punjab, India

Brucellosis is a neglected zoonosis that affects animals and people in much of the underdeveloped world. The disease is endemic in cattle in Punjab, India and controlling it is a public health challenge. Dairy farmers and farm labour commonly handle cattle faeces with bare hands and personal protective equipments are not used. No studies have been conducted about the shedding of Brucella species in faeces of sero positive cattle in the state. This study aimed to isolate and identify the Brucella species from faeces of sero positive cattle in Punjab, India. Faecal samples were collected from 350 Brucella sero positive cattle in Ludhiana district of Punjab, India. Isolation was performed using a pre-enriched Brucella selective broth medium as well as Brucella selective medium agar plates containing horse serum and Brucella selective supplements. Isolates were identified using Gram staining technique and rapid slide agglutination test, and then confirmed by using bcsp31 and 16s rRNA genus specific PCR. Isolates were further identified up to species level by using Bruce-Ladder multiplex PCR. Fourteen Brucella species were isolated, all of which showed coccobacilli on gram staining, positive rapid slide agglutination test and amplification of bcsp31 and 16s rRNA genes. Of the 14 isolates, 11 were identified as Brucella abortus and 3 were identified as Brucella melitensis. The study demonstrates that animal faeces could pose a potential risk for animal and human health and faeces of seropositive cattle must be handled with care.

Is There a Real Relationship between the Presence of Helicobacter pylori in Dental Plaque and Gastric Infection? A Genotyping and Restriction Fragment Length Polymorphism Study on Patient Specimens with Dyspepsia in Southwest Iran.

The oral cavity can act as an extra gastric reservoir for H. pylori, and the presence of the bacteria in the oral cavity is associated with a higher risk of dental caries development. This study aimed to determine the genotype and evaluate the association between the presence of H. pylori in dental plaque and gastric biopsy specimens in dyspeptic patients in Ahvaz, Southwest Iran. In this study, 106 patients with recruited dyspeptic complaints were selected, and from each patient, two gastric antral biopsy specimens and two dental plagues were examined. The presence of H. pylori was identified by the rapid urease test (RUT) and the amplification of ureAB and 16S rRNA genes. Also, to verify a hypothetical mouth-to-stomach infection route, the enzymatic digestions of three genes of cagA, vacA, and ureAB in H. pylori strains isolated from dental plaques and stomach samples were compared for each same case. H. pylori was found in the stomach of 52.8% (56/106) and the dental plaques of 17.9% (19/106) of the studied cases. On the other hand, H. pylori was recognized in the stomach of all 19 cases with oral colonization. Following a combination of restriction fragment lengths 21 polymorphism (RFLP) patterns of these three known genes on stomach and dental plague samples, 14 and 11 unique patterns were seen, respectively. However, for all H. pylori-positive cases (19), the comparison of RLFP patterns of these genes in dental plaque and gastric biopsy specimens was different for the same case. In this study, it seems that there is no significant association between the presence of H. pylori in dental plaque and the stomach of the same case.

Optimizing Fecal Occult Blood Test (FOBT) Colorectal Cancer Screening Using Gut Bacteriome as a Biomarker

BackgroundColorectal cancer (CRC) is a major cause of cancer mortality in the world. One of the most widely used screening tests for CRC is the immunochemical fecal occult blood test (iFOBT), which detects human hemoglobin from patient's stool sample. Although it is highly efficient in detecting blood from patients with gastro-intestinal lesions, such as polyps and cancers, the iFOBT has a high rate of false positive discovery. Recent studies suggested gut bacteria as a promising noninvasive biomarker for improving the diagnosis of CRC. In this study, we examined the composition of gut bacteria using iFOBT leftover from patients undergoing screening test along with a colonoscopy. MethodsAfter collecting data from more than 800 patients, we considered 4 groups for this study. The first and second groups were respectively “healthy” in which the patients had either no blood in their stool or had blood but no lesions. The third and fourth groups of patients had both blood in their stools with precancerous and cancerous lesions and considered either as low-grade and high-grade lesion groups, respectively. An amplification of 16S rRNA (V4 region) gene was performed, followed by sequencing along with various statistical and bioinformatic analysis. ResultsWe analyzed the composition of the gut bacteriome at phylum, class, genus, and species levels. Although members of the Firmicute phylum increased in the 3 groups compared to healthy patients, the phylum Actinobacteriota was found to decrease. Moreover, Blautia obeum and Anaerostipes hadrus from the phylum Firmicutes were increased and Collinsella aerofaciens from phylum Actinobacteriota was found decreased when healthy group is compared to the patients with high-grade lesions. Finally, among the 5 machine learning algorithms used to perform our analysis, both elastic net (AUC > 0.7) and random forest (AUC > 0.8) performs well in differentiating healthy patients from 3 other patient groups having blood in their stool. ConclusionOur study integrates the iFOBT screening tool with gut bacterial composition to improve the prediction of CRC lesions.

Black soldier fly (Hermetia illucens) larvae meal based extruded diets: potential to improve canine oral health

Abstract Black soldier fly larvae (BSFL) meal-based pet food is gaining traction in Europe. In order to unlock the full potential of antimicrobial peptides and fatty acids present in BSFL meal, we evaluated the role of BSFL meal containing pet food in modulating the oral health of dogs. During this study eight female beagle dogs were fed with two iso-nutritive diets in a cross-over design with two periods of 50 days each. These diets were made using defatted BSFL meal or low ash poultry by product (PBP) meal as the main protein source. At the end of the first period, the animals underwent a wash out period of 7 days and the treatments were inverted following this treatment. Oral health of dogs was measured by: (1) count of colony forming unit of volatile sulfur producing bacterial (VSC) in dental plaque; (2) DNA extraction and amplification of 16S rRNA genes from saliva for bacterial profiling; and (3) oral malodor scoring. We found significant reduction of VSC () before and after 50 days of BSFL based pet food consumption. Oral malodor score was not significantly different before and after BSFL based pet food consumption. However, there was a strong tendency for malodor reduction related to BSFL fed groups (). Finally, there was a significant difference () between treatments in the relative abundance of Moraxella, with higher abundance in dog’s saliva when fed BSFL diets. This bacterial group is identified as a marker of oral health. In conclusion, the consumption of BSFL meal-based diets has strong potential to decrease VSC and positively modulates the saliva microbiota, which can suppress the occurrence of halitosis and improve oral health.

Paratyphoid Salmonella serovars in chickens: molecular detection of virulence and antimicrobial resistance genes

Paratyphoid salmonellosis is a serious disease threatens the poultry industry worldwide, besides its public health hazard. The aims of this study were characterization of paratyphoid Salmonella spp. in chicken flocks of some Egyptian governorates, demonstration of the antimicrobial susceptibility of the isolated Salmonella spp., and detection of some virulence genes and antibiotic resistance genes using recent molecular techniques. A total of 238 organ samples were collected from 52 broiler, layer, and breeder chicken flocks, representing 9 Egyptian governorates. Conventional characterization of Salmonella isolates revealed a total isolation rate of 56.3% (134/238). Moreover, the isolation rates of Salmonella spp. were (49/79; 62%), (47/81; 58%), (10/18; 55.5%), (9/20; 45%), (2/6; 33.3%), (2/3; 66.7%), and (15/82; 53.6%) from liver, yolk sac, heart, spleen, caecum, ovary, and dead-in-shell embryos, respectively. A total of 32/238 (13.44%) isolates of Salmonella were found. Serological identification revealed presence of S. enteritidis (21.9%), S. kentucky (15.6%), S. typhimurium (12.5%), S. molade (12.5%), S. takoradi (9.4%), S. wingrove (6.3%), S. infantis (6.3%), S. tsevie (6.3), S. shangani (3.1%), S. bargny (3.1%), and S. papuana (3.1%). All Salmonella strains (32/32; 100%) were resistant to streptomycin, while almost all of them (31/32; 96.9%) were susceptible to meropenem. The amplification of 16S rRNA gene of Salmonella isolates using uniplex polymerase chain reaction (PCR) generated a specific Salmonella product of approximate 550 base pair. The multiplex PCR revealed presence of invA (100%), stn (65.6%), and sopB (40.6 %) virulence-associated genes as well as aadA1 (100%), blaTEM (59.4%), aadB (18.75%), and sul1 (28.1%) antibiotic resistance genes. In conclusion, virulent paratyphoid Salmonella spp. are circulating in the Egyptian flocks, causing economic loses. Additionally, they became resistant to the most commonly used field antibiotics. Therefore, regular molecular surveillance studies on the circulating Salmonella spp. and their resistance to the used antibiotics are of significant importance.

Preliminary molecular identification of proteolytic and lipolytic-enzyme producing bacteria isolated from sediment of Litopenaeus vannamei pond

The enzyme is a catalyst that serves as an accelerator in a biochemical process. A hydrolytic enzyme is among the existing enzymes useful for environmental organic waste degradation. This research aimed to (1) investigate the ability of bacteria isolated from Litopenaeus vannamei pond sediment to produce proteolytic and lipolytic enzymes, (2) identify the isolates with 16S rRNA gene amplification, and (3) construct a phylogenetic tree according to the 16S rRNA genes. The preliminary proteolytic assay consisted of a skim milk agar medium and the lipolytic assay consisted of a Tween 20/80 medium or so-called precipitation test. Among 28 bacterial isolates, 4 of them showed potential for proteolytic activity and 6 of them showed lipolytic activity as well. Further, the amplification of 16S rRNA gene showed that 5 out of the 28 isolates were closely related to Bacillus infantis SA 3.2 (IM8), Marinobacter koreensis SB 1.1 (IM6), Vibrio algynolyticus SA 4.2 (IM6), Streptomyces euryhalinus SB 1.2 (IM6), and Cytobacillus kochii SC 3.4 (IM6). Amongst the most active isolate, one of the was included in Actinobacteria phylum. Many Bacilli strains shows enzymatic activity such as amylase, protease and lypase. The genus Marinobacter also found to be able to produce hydrolytic enzyme such as amylase and protease. Furthermore, the genus Vibrio such as V. algynoliticus produce several enzymes i.e., gelatinase, lecithinase, caseinase, amylase and lipase. In summary, the bacteria from L. vannamei pond sediment exhibit a potential as proteolytic and lipolytic enzyme producers.

Seasonality, epidemiology and phylogeny of Theileria ovis with a note on hematological and biochemical changes in asymptomatic infected goats from Pakistan.

Caprine theileriosis, caused by Theileria ovis is a serious production issue, especially in the areas that depend on goats and sheep for milk, meat, and other economic benefits. Pakistan has a large goat population, but few reports have been documented from this country regarding PCR-based detection of T. ovis. The molecular prevalence of T. ovis, on a seasonal basis, in various goat breeds enrolled from Muzaffar Garh district of Punjab in Pakistan was determined from October 2018 to September 2019. In this study, 1084 goat blood samples were screened for the detection of T. ovis DNA through PCR-based amplification of 18S rRNA gene. Out of 1084 goats, 12 (1.11%) were infected with T. ovis. The parasite prevalence varied with the sampling seasons (Chi square test, P = 0.008), and the parasite prevalence was highest in goat blood samples collected in summer (2.39%) followed by winter (1.88%). DNA sequencing and BLAST analysis confirmed the presence of T. ovis, and the amplified isolates from the 18S rRNA gene of T. ovis were found to be highly conserved during phylogenetic analysis. Young goats (Fischer exact test, P = 0.022) were found more infected with T. ovis during the winter season. Infected goats had elevated white blood cell counts (Two-sample t-test, P = 0.04), blood urea nitrogen to Creatinine ratio (Two-sample t-test, P = 0.02) and decreased serum Creatinine (Two-sample t-test, P = 0.001) as compared to T. ovis negative goats. We report a relatively low molecular prevalence of T. ovis in goats from the Muzaffar Garh district. However, it is recommended that control measures to eradicate T. ovis infection in goats in this area should be taken.

Isolation, Identification and Characterization of Denitrifying Phototrophic Bacteria from Marine and Aquaculture Wastewater

Denitrifying bacteria occur primarily in the soil and play an important role in the nitrogen cycle and wastewater treatment. This study investigated the possibility of isolating denitrifying phototrophic bacteria from marine and aquaculture wastewater. The denitrifying ability of isolates was examined through their utilization of inorganic nitrogen and nitrate reductase test. The photosynthetic capability of the isolates was determined by detecting the photosynthetic pigments (bacteriochlorophyll and carotenoid). Molecular characterization of the isolates was carried out by the amplification of 16S rRNA gene. Forty-five different isolates were obtained and photosynthetic pigments were detected in 12 (8 from marine and 4 from aquaculture). Four of the isolates were found to grow on both inorganic nitrate and nitrite as the sole carbon source. Molecular characterization has shown that the isolates are denitrifying bacteria and the relationship between isolates and other denitrifying bacteria has been established by the construction of the phylogenetic tree. Hence denitrification and denitrifying bacteria can occur in both marine and aquaculture wastewaters.

Isolation and Characterization of Bacterial Endophytes from Small Nodules of Field-Grown Peanut.

It is evident that legume root nodules can accommodate rhizobial and non-rhizobial bacterial endophytes. Our recent nodule microbiome study in peanuts described that small nodules can harbor diverse bacterial endophytes. To understand their functional role, we isolated 87 indigenous endophytes from small nodules of field-grown peanut roots and characterized them at molecular, biochemical, and physiological levels. The amplified 16S rRNA genes and phylogenetic analysis of these isolates revealed a wide variety of microorganisms related to the genera Bacillus, Burkholderia, Enterobacter, Herbaspirillum, Mistsuaria, Pantoea, Pseudomonas, and Rhizobia. It was observed that 37% (100% identity) and 56% (>99% identity) of the isolates matched with the amplified sequence variants (ASVs) from our previous microbiome study. All of these isolates were tested for stress tolerance (high temperature, salinity, acidic pH) and phosphate (P) solubilization along with ammonia (NH3), indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate deaminase (ACCD), and siderophore production. The majority (78%) of the isolates were found to be halotolerant, thermotolerant, and acidophilic, and a few of them showed a significant positive response to the production of IAA, NH3, siderophore, ACCD, and P-solubilization. To evaluate the plant growth promotion (PGP) activity, plant and nodulation assays were performed in the growth chamber conditions for the selected isolates from both the non-rhizobial and rhizobial groups. However, these isolates appeared to be non-nodulating in the tested conditions. Nonetheless, the isolates 2 (Pantoea), 17 (Burkholderia), 21 (Herbaspirillum), 33o (Pseudomonas), and 77 (Rhizobium sp.) showed significant PGP activity in terms of biomass production. Our findings indicate that these isolates have potential for future biotechnological applications through the development of biologicals for sustainable crop improvement.