Metagenome-based microbial community analysis of urine-derived fertilizer.

Phosphorus is essential for food production and its supply is limited. Urine is an excellent source of phosphorus and one way to produce fertilizer is through conversion of urine to struvite (MgNH3PO4.6H2O). The present study aimed to understand the bacterial portion of the microbial community composition and dynamics of plasmid-mediated antimicrobial resistant genes during the optimized process of struvite production from composite human urine. Samples for DNA extraction was collected from fresh urine, stored urine and struvite during the process of struvite production. Shotgun metagenomic analysis was employed to understand the bacterial community. The most dominant phyla in the fresh and stored urine samples were Pseudomonadata, which comprised of 60% and 43% respectively, followed by Bacillota, comprised of 25% and 39% respectively. The struvite sample was dominated by the phylum Bacilliota (61%), Pseudomonadota (18%) and bacteroidota (12%). Members of the above phyla persisted in dominating each sample accordingly. Member of the family Morganellaceae was dominant in the fresh sample while the stored urine and struvite samples were dominated by the family Clostridiaceae. A decrease of members of the class Gammaproteobacteria was observed from the fresh to the struvite sample though not statistically significant. The genus Pseudomonas remained to be the most dominant member of Gammaproteobacteria in the fresh and stored urine sample with OTU count of 12,116 and 6,155 with a marked decrease by half in the stored sample. On the other hand, members of the genera Clostridium, Enterococcus, Bacteroides in the stored samples and Clostridium, Alkaliphilus and Pseudomonas in the struvite samples were dominant. 96% of the identified genera were shared in all the samples and the antimicrobial resistance genes (ARGs) identified in the fresh urine were shared by the struvite but not by the stored urine (e.g. sul, cat, aph and aac members). The presence of high abundance of ARGs in struvite needs attention in the persistence and transmissibility of the ARGs before application for agriculture.

Fate of antimicrobial resistance genes (ARG) and ARG carriers in struvite production process from human urine.

Struvite, a human urine-derived fertilizer types, is characterized by its low water solubility that renders it a slow-releasing eco-friendly fertilizer. Knowing the fate of antibiotic resistance genes in struvite is important since human urine carries microorganisms, viruses and mobilomes. In this study, urine samples were collected and struvite production was done using MgCl2. From the fresh, stored urine and struvite, DNA was extracted and metagenomic sequencing was done using Illumina HiSeqX. Metagenome-derived genome sequence analysis revealed the dominance of phages of Streptococcus, Bacillus and Escherichia, with nearly 50% abundance of streptococcus phage in fresh urine. Increased antibiotic resistance genes were found in the stored urine than in fresh and struvite samples. The top five resistance genes in all the three samples were to aminoglycosides, carbapenem, chloramphenicol, erythromycin and efflux pump, with key carrying pathogens including Acinetobacter, Aeromonas and Enterococcus. The identified families for carbapenem, aminoglycoside resistance and efflux pump were shown persistent in struvite with a shift in gene families. The detection of resistance-gene-laden mobilomes, including the last-resort antibiotics in the struvite sample, requires due attention before the implementation of struvite as fertilizer. Further optimization of the struvite production process with regard to the minimization of mobilomes is recommended.

Absence of significant association of trace elements in nails with urinary KIM-1 biomarker among residents of Addis Ababa in Upper Awash Basin, Ethiopia: a cross-sectional study.

The Akaki River in the Upper Awash Basin, which flows through Addis Ababa, the capital city of Ethiopia, has been highly polluted by sewage from factories and residential areas. A population-based cross-sectional study was used to assess the association between trace elements and kidney injury from residents living in polluted areas downstream (Akaki-Kality) versus upstream (Gullele) in Sub-Cities of Addis Ababa. A total of 95 individuals (53 from Akaki-Kality and 42 from Gullele) were included in the study. Kidney injury molecule 1 (KIM-1), lead, arsenic, cadmium, cobalt, lead, manganese, zinc, iron, copper, chromium and nickel were evaluated in residents' urine and nail samples. A large proportion (74%) of the sample population contained KIM-1, including 81% residents in Akaki-Kality and 64% residents in Gullele. KIM-1 was, however, not significantly different (p = 0.05) between the two Sub-Cities, with median of 0.224 ng/mL in Akaki-Kality and 0.152 ng/mL in Gullele. Most of the analyzed elements, except Pb, As, Cd and Co, were found in all of the nail samples, with median (µg/g) in the range of 442‒714 Fe, 97.0‒246 Zn, 11.6‒24.1 Mn, 4.49‒5.85 Cu, 1.46‒1.66 Cr and 1.22‒1.41 Ni. The high incidence of KIM-1 indicates a potential for long term renal tubular damage among residents of the Sub-Cities. The concentrations of the elements in nails were, however, not significantly associated (p = 0.05) with the corresponding levels of KIM-1 in urine. Hence, the observed KIM-1 might be related to exposure to toxic substances or factors other than those included in this study.

Trace Element Levels in Nails of Residents of Addis Ababa Are Shaped by Social Factors and Geography.

The Akaki catchment in Ethiopia is home to Addis Ababa and about five million people. Its watercourses receive a variety of wastes released by the residents and industries. River water is being used for irrigation, livestock watering, and other domestic purposes. This study tested the hypothesis that the river pollution would be reflected in higher levels of trace elements in the nails of residents living in Akaki-Kality Sub-City in the downstream, as compared to those living in Gullele Sub-City in the upstream of the Akaki catchment. Samples were taken and subsequently analysed for metals using inductively coupled plasma optical emission spectrometry (ICP-OES). The mean concentrations of Fe, Zn, Cu, Mn, Ni, Cr, Pb, and As in nails from Akaki-Kality were 488 ± 49, 106 ± 10, 5.2 ± 0.3, 13 ± 1.5, 11 ± 8, 2.2 ± 0.3, 0.09 ± 0.01, and 0.16 ± 0.01 µg/g, respectively. Likewise, the concentrations of Fe, Zn, Cu, Mn, Ni, Cr, Pb, and As in nails from Gullele were 1035 ± 135, 251 ± 10, 6.6 ± 0.4, 31 ± 3.7, 7.4 ± 1.7, 2.0 ± 0.3, 0.63 ± 0.01, and 0.25 ± 0.01 µg/g, respectively. Co and Cd were not detected. Contrary to the initial hypothesis, higher metal levels were found in nails of residents living in the upstream rather than the downstream area of the catchment. In particular, the concentrations of Fe (p = 0.000), Zn (p = 0.01), and Mn (p = 0.000) were significantly elevated in nails from Gullele and also high in comparison with internationally reported values. Besides, geography and social factors, especially education level, correlated to trace metals in nails. Most of the elements were significantly lower in the nails of individuals with a university degree compared to those who were illiterate or only completed primary school.

Urinary 8-OHdG level is not affected by geography and trace elements in nail of residents of Addis Ababa: It is shaped by interactions between different social factors.

The objective of this study was to evaluate the association between exposure to heavy metals and oxidative DNA damage among residents living in the potentially more polluted downstream (Akaki-Kality) area of Addis Ababa, in comparison to the upstream area (Gullele). For this, 8-hydroxy-2'-deoxyguanosine (8-OHdG) was used as a biomarker for oxidative DNA damage and heavy metals (Fe, Zn, Mn, Cu, Ni, Cr, Pb, As) as indicators of exposure. The concentrations of heavy metals in nails were determined using inductively coupled plasma optical emission spectroscopy (ICP-OES), and 8-OHdG in urine using Enzyme-Linked with Immunosorbent Assay (ELISA), from 95 residents of the two areas, upstream and downstream. The urinary 8-OHdG concentration was not significantly different (p = 0.05) between the two Sub-Cities, with mean of 18.50 ± 4.37 ng/mg Creatinine in Akaki-Kality and 17.30 ± 5.83 ng/mg Creatinine in Gullele. Also, there were no statistically significant (p = 0.05) difference among the different demographic groups according to gender, age, educational status, body mass index or habit of alcohol consumption. However, the interactions of sex with age, sex with alcohol consumption and alcohol consumption with education were found to affect the urinary 8-OHdG levels of residents of the two Sub-Cities. The mean concentrations (µg/g) of the elements were 488 and 1035 for Fe, 106 and 251 for Zn, 13.0 and 31.2 for Mn, 5.23 and 6.63 for Cu, 11.2 and 7.39 for Ni, 2.23 and 2.02 for Cr, 0.09 and 0.63 for Pb; and 0.16 and 0.25 for As, in nail samples from Akaki-Kality and Gullele, respectively. The determined concentrations of the heavy metals in nails were not significantly associated (p = 0.05) with the corresponding urinary levels of 8-OHdG. Hence, the observed 8-OHdG might have been caused by environmental exposure to toxic substances other than the analyzed heavy metals.

Evaluating tannery wastewater treatment performance based on physicochemical and microbiological characteristics: An Ethiopian case study.

Tanneries are an important industrial sector in Ethiopia; consequently, gaps in wastewater treatment process performance need to be identified as the country increases its emphasis on compliance. A case study was conducted to evaluate physicochemical and microbial water quality at a tannery near Addis Ababa. The treatment process was designed for the following: sulfide oxidation; biological oxygen demand reduction; and chromium removal. While some of Ethiopia's standards for industrial wastewater treatment were met through treatment, effluent COD, sulfide, total nitrogen, and total chromium guidelines were not. 16S rRNA gene analysis was used to evaluate the microbial community composition across the treatment train. The results show that common ruminant phyla were dominant throughout, with Firmicutes and Bacteroidetes comprising 77% to 82% relative abundance. The Firmicutes Clostridium increased consistently in relative abundance with treatment, comprising 39% to 61% of the total bacterial community in the effluent. Improved treatment is needed to meet environmental and public health goals. PRACTITIONER POINTS: Case Study of tannery wastewater treatment in Ethiopia shows ineffective treatment of chemical pollutants. Microbiological pollutants from tannery wastewater systems can introduce agents of importance to public health The microbiological composition of tannery influent, mixed liquor and effluent contains mostly four bacterial phyla lead by Firmicutes. Most pathogenic bacterial genera found in the tannery wastewater treatment system became a decreasing percentage of the total population. Clostridium comprises up to 61% of the effluent bacterial population and deserves further evaluation to better understand the consequences of its dominance.

Determination and health risk assessment of trace elements in the tap water of two Sub-Cities of Addis Ababa, Ethiopia.

Water is an essential component of all living things on earth and the contamination of water by heavy metals can cause detrimental health effects. This study aimed to determine the health risk posed by trace elements (Fe, Zn, Cu, Mn, Ni, Cr, Cd, Co, Pb, and As) present in the drinking water supplies of Gullele and Akaki-Kality Sub-Cities, upstream and downstream parts of Addis Ababa, respectively. The concentrations of the potentially toxic trace elements in the water samples were determined using inductively coupled plasma optical emission spectroscopy (ICP-OES). The highest concentration of the heavy metals was observed for Iron. Cadmium and cobalt were not detected in any of the tap water samples. Samples from Gullele contained higher levels of Fe and Mn, 220.3 ± 0.17 and 19.78 ± 0.08 µg/L, respectively compared to Akaki-Kality, 38.87 ± 0.14 and 2.08 ± 0.01 µg/L, respectively. Conversely, tap water from Akaki-Kality contained significantly higher levels of As than that from Gullele. Additionally, Cr and Ni were detected only in samples from Akaki-Kality, which might be due to the various industries in the area. The highest incremental lifetime cancer risk was found for arsenic, with values for children and adults in Akaki-Kality 2.50 × 10-4 and 4.50 × 10-4, respectively. Likewise, in Gullele Sub-City, it was 5.00 × 10-5 and 1.00 × 10-4 for adults and children, respectively. The results indicate that carcinogenic risk occurrence is probable from As in both studied areas.

Diagnostic Performance of SARS-CoV-2 IgM/IgG Rapid Test Kits for the Detection of the Novel Coronavirus in Ethiopia.

PURPOSE: Rapid severe acute respiratory syndrome coronavirus 2 test kits are crucial for bridging diagnostic gaps in health facilities and community screening mainly in resource limited settings. However, there is no objective evidence on their diagnostic performance. Thus, the study aimed to evaluate comparative diagnostic performance of three selected SARS-CoV-2 IgG/IgM rapid test kits in Ethiopia. METHODS: A cross-sectional study was conducted among 200 clients between May and July 2020 in Addis Ababa, Ethiopia. The performance of three SARS-CoV-2 rapid test kits EGENE, CTK BIOTECKs Onsite, and ACON Biotech were evaluated using blood specimens against RT-PCR on respiratory swabs. Sensitivity, specificity, and agreement with each other and to RT-PCR were computed using Vassarstats, MedCalc and SPSS version 23 statistical software. RESULTS: Test kits showed a heterogeneous comparative diagnostic performance in their sensitivity and specificity. The sensitivity was 61.18% (95% CI: 49.96-71.37%), 74.12% (95% CI: 63.28-82.74%) and 83.53% (95% CI: 73.57-90.38%) for kit A, B and C, respectively. Similarly, the specificity was 96.52% (90.81-98.88%), 94.78% (88.52-97.86%) and 94.78% (88.52-97.86%) for test kit A, B and C, respectively. The test kits have an agreement with RT-PCR with kappa value of 0.60 (0.48-0.83), 0.71 (0.65-0.93), and 0.80 (0.76-1.04) for A, B, and C, respectively. There was a significant difference on diagnostic performance among the three test kits and PCR with a p-value < 0.001 Cochran's Q test. CONCLUSION: The diagnostic performance of the test kits was promising and recommended for COVID-19 diagnostics in combination with RT-PCR to detect more infected patients. It allows determining the seroprevalence of the virus and true extent of SARS-COV-2 community spread in resource limited settings. We underline countries to evaluate rapid diagnostic test kits before diagnostic use.

A modified culture-based study of bacterial community composition in a tannery wastewater treatment plant.

Though culture-independent methods have been used in preference to traditional isolation techniques for characterization of microbial community of wastewater treatment plants, it is difficult to widely apply this approach in resource-poor countries. The present study aimed to develop a test to identify the culturable portion of bacterial community in a high-strength wastewater. Wastewater samples were collected from nitrification-denitrification and settling tanks of the treatment plant of Elmo Leather AB tannery located in Borås, Sweden. After cultivating on nutrient agar with the optimal dilution (10⁻²), phenotypic and biochemical identification of the bacteria were done with colony morphology, Gram reaction, growth on MacConkey, phenylethanol media, triple sugar Iron agar slants, catalase and oxidase tests. Biochemical grouping of the isolates was done based on their test results for MacConkey, phenylethanol media, triple sugar Iron agar and oxidase test reaction. From the biochemical groups, isolates were randomly selected for API test and 16SrRNA gene sequencing. The isolates from the denitrification, nitrification tank were identified to be Paracoccus denitrificans (67%), Azoarcus spp (3%) and Spingomonas wittichii (1%). From the settling tank, Paracoccus denitrificans (22%), Corynebacterium freneyi (20%) and Bacillus cereus (1%) were identified. The grouping based on biochemical test results as well as the identification based on sequencing has shown coherence except for discrepancies with the API test. The preliminary implications of the grouping based on culture-based characteristics and its potential application for resource-limited environmental microbial studies is discussed.

Antimicrobial susceptibility profile of selected Enterobacteriaceae in wastewater samples from health facilities, abattoir, downstream rivers and a WWTP in Addis Ababa, Ethiopia.

Background: Evaluation of antimicrobial susceptibility profile of various bacterial pathogens in the health facilities, abattoirs and related environment is important to assess potential risk of dissemination of resistant pathogens to the environment. There is limited information about antimicrobial susceptibility profile of common Enterobacteriaceae in waste water samples from hospitals, abattoirs and the downstream water bodies in Addis Ababa. The present study assessed antimicrobial susceptibility of bacteria belonging to the family Enterobacteriaceae isolated from wastewater samples (WWS) of two hospitals: Tikur Anbessa Specialized Hospital (TASH) and Minilik II hospital, a wastewater treatment plant (WWTP) and an abattoir, and downstream rivers in Addis Ababa. Results: A total of 54 bacterial isolates belonging to 6 species were identified: E.coli (32%), Salmonella 23%), Klebsiella pneumonia (15%), Enterobacter aerogenes (11%), Citrobacter (7%), Klebsiella oxytoca (6%) and Enterobacter cloacae (6%), respectively. Two strains of Citrobacter spp. isolated from TASH wastewater sample (WWS) were resistant to all 12 antimicrobials tested whereas an E. coli isolate from the same source was resistant to 11 antimicrobials. All isolates were resistant to 2 or more antimicrobials tested. Multi-drug resistance (MDR) to several antimicrobials was recorded, particularly in isolates obtained from hospital WWS and it was more common in Citrobacter and E. coli isolates. Extended spectrum betalactamase (ESBL) production was detected in 27.3% of MDR isolates, all of them obtained from hospital effluents whereas none of the isolates were carbapenemase producers. Conclusion: The present study revealed that Enterobacteriaceae in wastewater from hospitals, abattoir and downstream water bodies are resistant to commonly used antimicrobials. Hospital effluents contained more of MDR bacteria, posing significant public health threat through dissemination to the downstream water bodies.

Cytometric fingerprinting for analyzing microbial intracommunity structure variation and identifying subcommunity function.

Functions of complex natural microbial communities are realized by single cells that contribute differently to the overall performance of a community. Usually, molecular and, more recently, deep-sequencing techniques are used for detailed but resource-consuming phylogenetic or functional analyses of microbial communities. Here we present a method for analyzing dynamic community structures that rapidly detects functional (rather than phylogenetic) coherent subcommunities by monitoring changes in cell-specific and abiotic microenvironmental parameters. The protocol involves the use of flow cytometry to analyze elastic light scattering and fluorescent cell labeling, with subsequent determination of cell gate abundance and finally the creation of a cytometric community fingerprint. Abiotic parameter analysis data are correlated with the dynamic cytometric fingerprint to obtain a time-bound functional heat map. The map facilitates the identification of activity hot spots in communities, which can be further resolved by subsequent cell sorting of key subcommunities and concurrent phylogenetic analysis (terminal restriction fragment length polymorphism, tRFLP). The cytometric fingerprint information is based on gate template settings and the functional heat maps are created using an R script. Cytometric fingerprinting and evaluation can be accomplished in 1 d, and additional subcommunity composition information can be obtained in a further 6 d.