To Örebro University

Metal contamination of aquatic environments remains a major concern due to their persistence. The water flea Daphnia magna is an important model species for metal toxicity studies and water quality assessment. However, most research has focused on physiological endpoints such as mortality, growth, and reproduction in laboratory settings, as well as neglected toxicogenomic responses. Copper (Cu) and zinc (Zn) are essential trace elements that play crucial roles in many biological processes, including iron metabolism, connective tissue formation, neurotransmitter synthesis, DNA synthesis, and immune function. Excess amounts of these metals result in deviations from homeostasis and may induce toxic responses. In this study, we analyzed Daphnia magna transcriptomic responses to IC5 levels of Cu (120 µg/L) and Zn (300 µg/L) in environmental water obtained from a pristine lake with adjusted water hardness (150 mg/L CaCO3). The study was carried out to gain insights into the Cu and Zn regulated stress response mechanisms in Daphnia magna at transcriptome level. A total of 2,688 and 3,080 genes were found to be differentially expressed (DEG) between the control and Cu and the control and Zn, respectively. There were 1,793 differentially expressed genes in common for both Cu and Zn, whereas the number of unique DEGs for Cu and Zn were 895 and 1,287, respectively. Gene ontology and KEGG pathways enrichment were carried out to identify the molecular functions and biological processes affected by metal exposures. In addition to well-known biomarkers, novel targets for metal toxicity screening at the genomic level were identified.

Biocide resistance poses a significant challenge in industrial processes, with bacteria like Pseudomonas oleovorans exhibiting intrinsic resistance to traditional antimicrobial agents. In this study, the impact of biocide exposure on the metabolome of two P. oleovorans strains, namely, P. oleovorans P4A, isolated from contaminated coating material, and P. oleovorans 1045 reference strain, were investigated. The strains were exposed to 2-Methylisothiazol-3(2H)-one (MI) MIT, 1,2-Benzisothiazol-3(2H)-one (BIT), and 5-chloro-2-methyl-isothiazol-3-one (CMIT) at two different sub-inhibitory concentrations and the lipids and polar and semipolar metabolites were analyzed by ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry UPLC-Q-TOF/MS. Exposure to the BIT biocide induced significant metabolic modifications in P. oleovorans. Notable changes were observed in lipid and metabolite profiles, particularly in phospholipids, amino acid metabolism, and pathways related to stress response and adaptation. The 1045 strain showed more pronounced metabolic alterations than the P4A strain, suggesting potential implications for lipid, amino acid metabolism, energy metabolism, and stress adaptation. Improving our understanding of how different substances interact with bacteria is crucial for making antimicrobial chemicals more effective and addressing the challenges of resistance. We observed that different biocides trigged significantly different metabolic responses in these strains. Our study shows that metabolomics can be used as a tool for the investigation of metabolic mechanisms underlying biocide resistance, and thus in the development of targeted biocides. This in turn can have implications in combating biocide resistance in bacteria such as P. oleovorans.

Metal contamination of aquatic environments remains a major concern and has received significant attention in recent years. The present study aimed to evaluate the effects of metal mixtures of varying concentrations over time in a lake receiving runoff water from a decommissioned mine. By subjecting several organisms to this water, we aimed to identify the most susceptible species, thus enabling a comprehensive evaluation of the risk posed by different toxins to the biotic environment.

We have evaluated the effects of mixed metal exposure on survival and stress gene expression in selected invertebrate and vertebrate model species. Our observations revealed differences in sensitivity among the invertebrate models Caenorhabditis elegans, Daphnia magna, Ceriodaphnia dubia, and Heterocypris incongruens, as well as in the vertebrate model Zebrafish (Danio rerio) and two cell lines; a zebrafish liver cell line (ZFL) and a human hepatocellular carcinoma cell line (HepG2). While the sensitivity shows great variation among the tested species, the expression of metallothionein was consistent with the levels of metals found in the mixed exposure media. Despite differences in acute toxicity, the universal induction of mt1/A and mt2/B genes make them an important biomarker for assessing the environmental risk of metals.

BACKGROUND: Diarrhea is a serious health problem in children, with the highest mortality rate in sub-Saharan Africa. Diarrheagenic Escherichia coli (DEC) is among the major bacterial causes of diarrhea in children under age five. The present study aims to determine molecular epidemiology and antimicrobial resistance profiles of DEC and identify contributing factors for acquisition among children under age five in Central Ethiopia.

METHODS: A health facility-centered cross-sectional study was conducted in Addis Ababa and Debre Berhan, Ethiopia, from December 2020 to August 2021. A total of 476 specimens, 391 from diarrheic and 85 from non-diarrheic children under age five were collected. Bacterial isolation and identification, antimicrobial susceptibility, and pathotype determination using polymerase chain reaction (PCR) were done.

RESULTS: Of the 476 specimens analyzed, 89.9% (428/476) were positive for E. coli, of which 183 were positive for one or more genes coding DEC pathotypes. The overall prevalence of the DEC pathotype was 38.2% (183/476). The predominant DEC pathotype was enteroaggregative E. coli (EAEC) (41.5%, 76/183), followed by enterotoxigenic E. coli (21.3%, 39/183), enteropathogenic E. coli (15.3%, 28/183), enteroinvasive E. coli (12.6%, 23/183), hybrid strains (7.1%, 13/183), Shiga toxin-producing E. coli (1.6%, 3/183), and diffusely-adherent E. coli (0.6%, 1/183). DEC was detected in 40.7% (159/391) of diarrheic and 28.2% (24/85) in non-diarrheic children (p = 0.020). The majority of the DEC pathotypes were resistant to ampicillin (95.1%, 174/183) and tetracycline (91.3%, 167/183). A higher rate of resistance to trimethoprim-sulfamethoxazole (58%, 44/76), ciprofloxacin (22%, 17/76), ceftazidime and cefotaxime (20%, 15/76) was seen among EAEC pathotypes. Multidrug resistance (MDR) was detected in 43.2% (79/183) of the pathotypes, whereas extended spectrum ß-lactamase and carbapenemase producers were 16.4% (30/183) and 2.2% (4/183), respectively.

CONCLUSION: All six common DEC pathotypes that have the potential to cause severe diarrheal outbreaks were found in children in the study area; the dominant one being EAEC with a high rate of MDR.

Per- and polyfluoroalkyl substances (PFAS) are anthropogenic compounds threatening water quality and food safety worldwide. Phytoremediation is a nature-based, cost-effective, and scalable solution with high potential for treating PFAS-contaminated sites. However, there is a large knowledge gap regarding choice of plant species and methods to enhance performance. This study assessed the PFAS phytoextraction potential of sunflower (Helianthus annuus), mustard (Brassica juncea), and industrial hemp (Cannabis sativa) in a greenhouse experiment, using inorganic fertilizer and a microbial mixture as supplements. PFAS concentrations were measured using UPLC-MS/MS, and bioconcentration factors for different plant tissues and removal efficiency were determined. Perfluoroalkyl carboxylic acid (PFCA) accumulation was 0.4-360 times higher than that of perfluoroalkyl sulfonic acid (PFSA) homologues of similar perfluorocarbon chain length. Inorganic fertilizer significantly (p < 0.001) reduced PFAS concentration in all plant tissues, whereas the microbial mixture tested did not affect PFAS concentration. PFAS uptake ranged from 0.2 to 33% per crop cycle. Overall, the potential number of crop cycles required for removal of 90% of individual PFAS ranged from six (PFPeA) to 232 (PFOA) using sunflower, 15 (PFPeA) to 466 (PFOS) using mustard and nine (PFPeA) to 420 (PFOS) using Hemp. In this study, the percentage of PFAS removal by plants was determined, and an estimation of the time required for PFAS phytoextraction was determined for the first time. This information is important for practical phytoremediation applications.

Zinc (Zn) is an essential element that influences many cellular functions. Depending on bioavailability, Zn can cause both deficiency and toxicity. Zn bioavailability is influenced by water hardness. Therefore, water quality analysis for health-risk assessment should consider both Zn concentration and water hardness. However, exposure media selection for traditional toxicology tests are set to defined hardness levels and do not represent the diverse water chemistry compositions observed in nature. Moreover, these tests commonly use whole organism endpoints, such as survival and reproduction, which require high numbers of test animals and are labor intensive. Gene expression stands out as a promising alternative to provide insight into molecular events that can be used for risk assessment. In this work, we apply machine learning techniques to classify the Zn concentrations and water hardness from Daphnia magna gene expression by using quantitative PCR. A method for gene ranking was explored using techniques from game theory, namely, Shapley values. The results show that standard machine learning classifiers can classify both Zn concentration and water hardness simultaneously, and that Shapley values are a versatile and useful alternative for gene ranking that can provide insight about the importance of individual genes.

The spread of antimicrobial-resistant pathogens is a global health concern. Most studies report high levels of antimicrobial resistance genes (ARGs) in the aquatic environment; however, levels associated with sediments are limited. This study aimed to investigate the distribution of ARGs in the sediments and water of the Akaki river in Addis Ababa, Ethiopia. The diversity and abundance of 84 ARGs and 116 clinically important bacteria were evaluated from the sediments and water collected from five sites in the Akaki river. Most of the ARGs were found in the city close to anthropogenic activities. Water samples collected in the middle catchment of the river contained 71-75% of targeted ARGs, with genes encoding aminoglycoside acetyltransferase (aac(6)-Ib-cr), aminoglycoside adenylyl transferase (aadA1), β-lactamase (bla_OXA-10)_, quinolone resistance S (qnrS), macrolide efflux protein A (mefA), and tetracycline resistance (tetA), were detected at all sampling sites. Much fewer ARGs were detected in all sediments, and those near the hospitals had the highest diversity and level. Despite the lower levels and diversity, there were no unique ARGs detected in the sediments that were also not detected in the waters. A wide range of clinically relevant pathogens were also detected in the Akaki river. The findings suggest that the water phase, rather than the sediments in the Akaki river, is a potential conduit for the spread of ARGs and antibiotic-resistant bacteria.

Ceftazidime-avibactam and ceftolozane-tazobactam are approved for the treatment of complicated Gram-negative bacterial infections including multidrug-resistant (MDR) Pseudomonas aeruginosa. Resistance to both agents has been reported, but the underlying mechanisms have not been fully explored. This study aimed to correlate β-lactamases with phenotypic resistance to ceftazidime-avibactam and/or ceftolozane-tazobactam in MDR-P. aeruginosa from Qatar. A total of 525 MDR-P. aeruginosa isolates were collected from clinical specimens between 2014 and 2017. Identification and antimicrobial susceptibility were performed by the BD Phoenix^TM system and gradient MIC test strips. Of the 75 sequenced MDR isolates, 35 (47%) were considered as having difficult-to-treat resistance, and 42 were resistant to ceftazidime-avibactam (37, 49.3%), and/or ceftolozane-tazobactam (40, 53.3%). They belonged to 12 sequence types, with ST235 being predominant (38%). Most isolates (97.6%) carried one or more β-lactamase genes, with bla_OXA-488 (19%) and bla_VEB-9 (45.2%) being predominant. A strong association was detected between class B β-lactamase genes and both ceftazidime-avibactam and ceftolozane-tazobactam resistance, while class A genes were associated with ceftolozane-tazobactam resistance. Co-resistance to ceftazidime-avibactam and ceftolozane-tazobactam correlated with the presence of bla_VEB-9, bla_PDC-35, bla_VIM-2, bla_OXA-10 and bla_OXA-488. MDR-P. aeruginosa isolates resistant to both combination drugs were associated with class B β-lactamases (bla_VIM-2) and class D β-lactamases (bla_OXA-10), while ceftolozane-tazobactam resistance was associated with class A (bla_VEB-9), class C (bla_VPDC-35), and class D β-lactamases (bla_OXA-488).

Carbapenemase-producing Aeromonas species are an emerging health threat. This study aimed to determine carbapenemase-mediated resistance among Aeromonas isolates from the Akaki river, Ethiopia during the dry and wet seasons in 2019-2020. Antimicrobial susceptibility to carbapenems and cephalosporins was determined and carbapenemase production was confirmed. Of 163 isolates, the majority were human pathogens Aeromonas caviae (62), Aeromonas hydrophila (33) and Aeromonas veronii (49). These isolates were resistant to carbapenem and cephalosporin antibiotics, with the highest resistance to cefotaxime 86 (59.7%), ertapenem 71 (49.3%) and imipenem 65 (45.1%). Resistance to carbapenem antibiotics varied between species, where most A. veronii 37 (75.5%) and A. hydrophila 28 (84.8%) were resistant to imipenem, whereas all A.caviae were sensitive. A. veronii, A. caviae and A. hydrophila resistance to meropenem was 31 (63.3%), 3 (4.8%) and 19 (57.6%), respectively. Of isolates resistant to carbapenem, 82.1% A. hydrophila and 94.4% A. veronii were carbapenemase producers. Cephalosporin resistance also varied among the different species. The highest resistance to carbapenem antibiotics was in isolates collected during the wet season (p<0.05); however, it was not consistent across all classes of antibiotics tested. The rivers in megacities could be reservoirs of carbapenemase-producing Aeromonas spp.

Escherichia coli toxins are the most common types found in association with disease in E. coli bacteria. They are found in nearly all pathogenic E. coli bacteria either released from the bacteria or E. coli cell or both transmitted via the orofecal route. The present study evaluated the relationship between E. coli toxins detected in ‘improved’ drinking water sources and toxins found in stool samples from individuals with diarrhea that consumed the water. Improved water is defined as the water source, by the nature of their construction or through active intervention, are protected from external contamination, particularly with fecal matter. A correlation between toxins in the water with toxins in the stools suggests that contaminated drinking water is a potential infection source. Stool samples were collected from people with diarrhea (248 stool samples) and their drinking water at their home (248 water samples). Only diarrhea patients that reported use of improved drinking water sources as per the definition of the WHO/UNICEF Joint Monitoring Programme were included in the study. E. coli isolates were analysed for 5 major toxins (enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1), heat-stable enterotoxin a (Sta); shiga-like toxin 1 (Stx1), shiga-like toxin 2 (Stx2), and heat-liable enterotoxin (LT)) using a multiplex polymerase chain reaction (mPCR). Descriptive statistics, Chi-square (χ2) test, Fisher's exact, logistic regression, and bivariate Pearson correlation coefficients were used to examine the association and correlation of toxins in the drinking water with those in the stool samples. Of the 248 households, 24% had positive results in both water and stool samples, 63% [Confidence Interval (CI): 55–67%] had positive results in water samples only and 46% [CI: 37- 49%] were found to be positive for toxins in stool samples only. EAST1 was the most frequently detected from the 5 toxins evaluated, with 33% in the water and 38% in the stool. ESAT1, Stx1, and LT toxins were the most commonly found contaminants in water from improved water sources. The prevalence of toxins in household water samples was higher than that obtained from yard connections (51%) and piped systems connected to public stands (42%), compared to the other types of water sources. Both a positive correlation (P < 0.05; r = 0.412) and statistically significant association (P=0.0001) was found between the toxins in the water and toxins in the stool. EAST1 in the stool was significantly correlated with overall toxins in water (r = 0.378) and increased the probability of the occurrence of EAST1 in water (odds ratio (OR)=4.96, 95% CI, 2.81–8.74; P=0.000). E. coli toxins have been found to be effective biomarkers for detecting possible drinking water contamination in households with diarrhea. Long-term planning for the protection and/or treatment of stored household drinking water to ensure toxins free water at the point of use are required.