Morphological, Biochemical and Genotypic Analysis of Zoonotic Campylobacter jejuni Isolated from Chicken Meat Samples

Background: Campylobacter species are a leading cause of most important food-borne diarrhoeal illness worldwide while, poultry has been identified as a significant cause of Campylobacter infection in humans. C. jejuni is highly effective in colonizing chicken intestinal mucosa without causing any clinical manifestations and the consumption of poultry meat is the major source of transmission of bacteria to humans. Methods: The total of 19 chicken meat samples collected from retail markets in Chennai were screened by cultural examination, further subjected to phenotypic characterization using biochemical test and genotypic characterization using polymerase chain reaction assay targeting hip O and map A genes. Result: All the isolates showed growth on modified blood free charcoal cefoperazone deoxycholate agar media (mCCDA) and 18 (94.73%) samples showed typical morphological characteristics. The 12 (63.15%) isolates showed biochemical reactions positive. The results from polymerase chain reaction showed that 10 (83.33%) isolates were positive for C. jejuni. This study suggested that, it is essential to investigate the incidence of Campylobacter jejuni infection in poultry and the risk factors at all production stages of meat production to help reducing the disease in humans in terms of food safety.

Genomic characteristics and comparative genomics of Salmonella enterica subsp. enterica serovar Schwarzengrund strain S16 isolated from chicken feces

Background Salmonella enterica subsp. enterica serovar Schwarzengrund (S. Schwarzengrund) is most frequently isolated from commensals humans or poultry. Here we report S. Schwarzengrund strain S16, the first sequenced genome in the Republic of Korea. Additionally, genome sequencing for strain S16 was performed and compared with other S. Schwarzengrund genomes obtained from public database. Results Strain S16 was isolated from chicken feces. The complete genome consists of one chromosome and one plasmid. The genome size is 4,822,755 bp with 4852 coding sequences. Strain S16 was determined as serovar Schwarzengrund by in silico serotyping and typed as sequence type (ST) 96. Forty-six S. Schwarzengrund genomes yielded a pangenome of 7112 genes, core-genome of 3374 genes, accessory-genome of 2906 genes, and unique-genome of 835 genes. Eighty-one genes were unique to strain S16, including hypothetical proteins and transcriptional regulators. Genotypic analysis of antibiotic resistance of strain S16 confirmed resistance to amikacin, ciprofloxacin, sulfamethoxazole, streptomycin, and tetracycline. Unlike other S. Schwarzengrund genomes, strain S16 had a mutation of gyrB. Moreover, similar to other S. Schwarzengrund genomes reported in other countries, strain S16 was harbored for 153 virulence genes including Saf operon and cdtB gene. All the antibiotic resistance genes and virulence genes were present in the core- or accessory-genomes. Conclusions Complete genome of strain S16 was sequenced. Comparative genomic analysis revealed several genes responsible for antibiotic resistance and specific genomic features of strain S16 and identified virulence factors that might contribute to the human and animal pathogenicity of other S. Schwarzengrund genomes.

Prevalence and Risk Factors of Kaposi’s Sarcoma-Associated Herpesvirus Infection among Han and Uygur Populations in Xinjiang, China

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS), which is endangering human health worldwide, especially in Africa, Europe, the United States, and parts of Asia. The aim of this study was to investigate the prevalence of KSHV in Xinjiang. Three KSHV recombinant proteins (ORF65, ORF73, and K8.1) were used to detect KSHV infection. The serum samples to be tested were detected by an indirect ELISA method. The overall infection rate of KSHV in Xinjiang was 25.60%, with a higher infection rate in the Uygur population of 29.79%. After adjusting for possible confounders, Uygur (OR = 3.95, 95% CI 2.64–6.12, P < 0.001 ), agriculture and livestock (OR = 1.60, 95% CI 1.20–2.17, P = 0.002), age ≤ 50 years (OR = 1.50, 95% CI 1.13–2.00, P = 0.006), and predominantly meat-based diet (OR = 1.72, 95% CI 1.11–2.78, P = 0.018) were significantly associated with the odds of KSHV seropositivity correlation. Three unique sequences of KSHV were obtained in this study; genotypic analysis showed that the three unique sequences were all subtype A2.

Rice backcross population assessment for iron tolerance through phenotypic and genotypic analyses

Iron toxicity has become a serious issue affecting rice (Oryza sativa L.) production in many irrigated lowland areas. The selection of Fe2+-tolerant rice cultivars under iron toxicity conditions and the identification of molecular markers are good approaches to obtaining tangible results. This study aimed to identify simple sequence repeat (SSR) markers that were associated with iron tolerance traits in a rice backcross population. A total of 117 seedlings from the backcross (BC3F2) of ‘OM6830’/‘AS996’//‘AS996’ were phenotyped at the 4-week-seedling stage at Ton Duc Thang University, Ho Chi Minh City, Vietnam. The rice population was screened in Yoshida nutrient medium supplemented with FeCl2 at a concentration of 150 mg L−1 under greenhouse conditions. Phenotypic analysis was conducted by scoring two parameters, namely, root length and leaf bronzing. Genotypic analysis was carried out on the BC3F2 population by using four markers, i.e., RM6, RM240, RM252, and RM451, for association analysis with iron tolerance. A total of 23 BC3F2 lines were selected on the basis of their higher tolerance (score 1) for Fe2+ compared with the tolerant parental line ‘AS996’. The markers RM6 and RM240 were highly polymorphic and identified different Fe2+-tolerant lines in the BC3F2 population. Among the BC3F3 progeny derived from the selected 23 BC3F2 lines, BC3F3-7 was identified as the most Fe2+-tolerant line. BC3F3-15 was also found to be Fe2+ tolerant. Both lines showed good development capability and provided high yields under stress conditions. These tolerant BC3F3 lines could be further screened with additional SSR markers in future breeding programs aiming to increase rice production in iron-contaminated areas of the Mekong Delta, Vietnam.

Frateuria flava sp. nov., isolated from soil

A Gram-stain-negative, aerobic, rod-shaped and non-motile novel bacterial strain, designated MAH-13T, was isolated from a soil sample. The colonies were observed to be yellow-coloured, smooth, spherical and 1.8–3.0 mm in diameter when grown on nutrient agar medium for 2 days. Strain MAH-13T was found to be able to grow at 20–40 °C, at pH 5.0–10.0 and with 0–1.0% NaCl (w/v). Cell growth occurred on tryptone soya agar, Luria–Bertani agar, nutrient agar and Reasoner's 2A agar. The strain was found to be positive for both oxidase and catalase tests. The strain was positive for hydrolysis of casein, starch, DNA and l-tyrosine. According to 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Frateuria and to be closely related to Frateuria terrea DSM 26515T (98.2% similarity), Dyella thiooxydans ATSB10T (98.2 %), Frateuria defendens HyOGT (97.9 %), Rhodanobacter glycinis MO64T (97.8 %) and Frateuria aurantia DSM 6220T (97.8 %). The novel strain MAH-13T has a draft genome size of 3 682 848 bp (40 contigs), annotated with 3172 protein-coding genes, 49 tRNA genes and three rRNA genes. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain MAH-13T and five closely related type strains were in the range of 73.7–85.5 % and 20.7–30.1%, respectively. The genomic DNA G+C content was determined to be 68.0 mol%. The predominant isoprenoid quinone was ubiquinone 8. The major fatty acids were identified as iso-C15:0, iso-C16:0 and summed feature 9 (iso-C17 : 1 ω9c and/or C16:0 10-methyl). On the basis of dDDH and ANI values, genotypic analysis, and chemotaxonomic and physiological data, strain MAH-13T represents a novel species within the genus Frateuria , for which the name Frateuria flava sp. nov. is proposed, with MAH-13T (=KACC 19743T=CGMCC 1.13655T) as the type strain.

Mitochondrial Transfer in Saccharomyces cerevisiae

<p>This thesis investigated mitochondrial transfer in Saccharomyces cerevisiae, between respiratory compromised B18p⁰ recipient and respiratory competent donor cells. The respiratory compromised strain had three red fluorescent proteins tagged to the membrane, nucleus and cytoplasm (triple RFP-B18p⁰) and is referred to as the B18p⁰ strain. B18p⁰ cells did not contain mitochondrial DNA, causing it to be respiratory compromised and required a fermentable carbon source, such as glucose/dextrose, for proliferation. The respiratory competent strain used had a green fluorescent protein tagged to the Tom70 mitochondrial protein (Tom70-GFP) and is referred to as the Tom70 strain. The Tom70 cells contained the nuclear encoded URA3 cassette, allowing for negative selectivity of this strain using 5-FOA. S. cerevisiae strains were co-cultured together in media containing only non-fermentable carbon sources (YPGE), plated on YPGE plates containing 5-FOA and colonies grown were distinguished post-co-culture based on their distinct phenotypic and genotypic characteristics. Fluorescent analysis of co-culture colonies revealed the presence of 5-FOA resistant Tom70 cells and some red B18p⁰ cells that had acquired the ability to grow on non-fermentable carbon sources. Genotypic analysis revealed that the majority of these red colonies had acquired mtDNA as well as the nuclear encoded, Tom70 specific URA3 cassette. Several permutations of co-cultures were performed, using different ratios of recipient and donor cells and single-gene deletion donor cells. Purified mitochondria from Tom70 cells were tried to be transferred into B18p⁰ cells using centrifugation forces to induce a higher occurrence frequency of mitochondrial transfer. Metabolic support experiments were conducted to investigate if the Tom70 strain could provide metabolic support to the B18p⁰ strain without mitochondrial transfer. Results indicate that no permutation induced potential mitochondrial transfer at a higher rate than others. However, results indicate that mitochondrial transfer did occur at low frequencies, potentially through the fusion of respiratory competent and respiratory compromised cells. Forced transfer did not increase the occurrence frequency of B18p⁰ cells to take up mitochondria and Tom70 cells did not provide metabolic support to B18p⁰ cells.</p>

Mitochondrial Transfer in Saccharomyces cerevisiae

<p>This thesis investigated mitochondrial transfer in Saccharomyces cerevisiae, between respiratory compromised B18p⁰ recipient and respiratory competent donor cells. The respiratory compromised strain had three red fluorescent proteins tagged to the membrane, nucleus and cytoplasm (triple RFP-B18p⁰) and is referred to as the B18p⁰ strain. B18p⁰ cells did not contain mitochondrial DNA, causing it to be respiratory compromised and required a fermentable carbon source, such as glucose/dextrose, for proliferation. The respiratory competent strain used had a green fluorescent protein tagged to the Tom70 mitochondrial protein (Tom70-GFP) and is referred to as the Tom70 strain. The Tom70 cells contained the nuclear encoded URA3 cassette, allowing for negative selectivity of this strain using 5-FOA. S. cerevisiae strains were co-cultured together in media containing only non-fermentable carbon sources (YPGE), plated on YPGE plates containing 5-FOA and colonies grown were distinguished post-co-culture based on their distinct phenotypic and genotypic characteristics. Fluorescent analysis of co-culture colonies revealed the presence of 5-FOA resistant Tom70 cells and some red B18p⁰ cells that had acquired the ability to grow on non-fermentable carbon sources. Genotypic analysis revealed that the majority of these red colonies had acquired mtDNA as well as the nuclear encoded, Tom70 specific URA3 cassette. Several permutations of co-cultures were performed, using different ratios of recipient and donor cells and single-gene deletion donor cells. Purified mitochondria from Tom70 cells were tried to be transferred into B18p⁰ cells using centrifugation forces to induce a higher occurrence frequency of mitochondrial transfer. Metabolic support experiments were conducted to investigate if the Tom70 strain could provide metabolic support to the B18p⁰ strain without mitochondrial transfer. Results indicate that no permutation induced potential mitochondrial transfer at a higher rate than others. However, results indicate that mitochondrial transfer did occur at low frequencies, potentially through the fusion of respiratory competent and respiratory compromised cells. Forced transfer did not increase the occurrence frequency of B18p⁰ cells to take up mitochondria and Tom70 cells did not provide metabolic support to B18p⁰ cells.</p>

Molecular Epidemiology of Hepatitis B Virus Among HIV Co-Infected and Mono-Infected Cohorts in Northwest Ethiopia

Background Hepatitis B virus (HBV) infection is a particular concern in human immunodeficiency virus (HIV) infected individuals. In Ethiopia, detailed clinical and virological descriptions of HBV prevailing during HIV co-infection and symptomatic liver disease patients are lacking. The aim of this study was to investigate HBV virological characteristics from Ethiopian HBV/HIV co-infected and HBV mono-infected individuals. Methods A total of 4105 sera from HIV positive individuals, liver disease patients, and blood donors were screened serologically for HBV. The overlapping polymerase/surface genome region of HBV from 180 infected individuals was extracted, amplified, and sequenced for genotypic analysis. Results The HBsAg seroprevalence was detected 43% in liver disease patients, 8.4% in blood donors, and 6.7% in HIV/HBV co-infected individuals. The occult HBV prevalence was 3.7% in HIV/HBV co-infected individuals and 2.8% in blood donors with an overall prevalence rate of 3.4%. A phylogenetic analysis showed three HBV genotypes; A (61.1%), D (38.3%) and E (0.6%). Genotype A belongs to subtypes A1 (99.1%) and A9 (0.9%), but genotype D showed heterogeneous subtypes; D2 (63.8%) followed by D4 (21.7%), D1 (8.7%), D3 (4.3%), and D10 (1.4%). Conclusions The HIV/HBV co-infected individuals and blood donors showed lower HBsAg seroprevalence compared to liver diseases patients. Occult HBV prevalence showed no difference between HIV/HBV co-infected and blood donor groups. This study demonstrated predominance distribution of HBV subtypes A1 and D2 in northwest Ethiopia. The observed virological characteristics could contribute for evidence-based management of viral hepatitis in Ethiopia where antiretroviral therapy guidelines do not cater for viral hepatitis screening during HIV co-infection.

Clinical and Genetic Aspects of Juvenile Onset Pompe Disease

Little is known about clinical symptomatology and genetics of juvenile onset Pompe disease (JOPD). The aims of this study were to analyze how these children are diagnosed, what clinical problems they have, and how phenotype is related to genotype. To accomplish this, we analyzed retrospectively data of 34 patients diagnosed after their first and before completion of their 18th birthday. Median age at diagnosis was 3.9 (range 1.1–17) years. Eight patients (23.5%) developed initial symptoms in the first year, 12 (35%) between 1 and 7 years, and 6 (18%) thereafter. Eight (23.5%) had no clinical symptoms at the time of diagnosis. Indications for diagnostics were a positive family history in three (9%), hyperCKemia in eight (23.5%), motor developmental delay in three (9%), and muscle weakness and/or pain in 17 (50%). Rare clinical signs were failure to thrive, recurrent diarrhea, and suspected hepatopathy with glycogen storage. Thirty-two different mutations were identified. Twenty-seven patients (79.5%) carried the milder c.32–13T > G mutation, known to be associated with a broad range of phenotypes. Three out of eight patients manifesting within the first year of life showed generalized muscle weakness, hypertrophic cardiomyopathy, and had to be ventilated during the course of disease, thereby demonstrating clinical overlap with infantile onset Pompe disease.These findings demonstrate that the phenotype of JOPD is broad and that the differential is not only restricted to neuromuscular disorders. Genotypic analysis was useful to delineate subjects with early onset JOPD from those with IOPD, but overall genotype–phenotype correlation was poor.