Phylogenetic Analysis of Transmission Dynamics of Dengue in Large and Small Population Centers, Northern Ecuador.

Although dengue is typically considered an urban disease, rural communities are also at high risk. To clarify dynamics of dengue virus (DENV) transmission in settings with characteristics generally considered rural (e.g., lower population density, remoteness), we conducted a phylogenetic analysis in 6 communities in northwestern Ecuador. DENV RNA was detected by PCR in 121/488 serum samples collected from febrile case-patients during 2019-2021. Phylogenetic analysis of 27 samples from Ecuador and other countries in South America confirmed that DENV-1 circulated during May 2019-March 2020 and DENV-2 circulated during December 2020-July 2021. Combining locality and isolation dates, we found strong evidence that DENV entered Ecuador through the northern province of Esmeraldas. Phylogenetic patterns suggest that, within this province, communities with larger populations and commercial centers were more often the source of DENV but that smaller, remote communities also play a role in regional transmission dynamics.

Risk factors for extended-spectrum beta-lactamase (ESBL)-producing E. coli carriage among children in a food animal-producing region of Ecuador: A repeated measures observational study.

BACKGROUND: The spread of antibiotic-resistant bacteria may be driven by human-animal-environment interactions, especially in regions with limited restrictions on antibiotic use, widespread food animal production, and free-roaming domestic animals. In this study, we aimed to identify risk factors related to commercial food animal production, small-scale or "backyard" food animal production, domestic animal ownership, and practices related to animal handling, waste disposal, and antibiotic use in Ecuadorian communities. METHODS AND FINDINGS: We conducted a repeated measures study from 2018 to 2021 in 7 semirural parishes of Quito, Ecuador to identify determinants of third-generation cephalosporin-resistant E. coli (3GCR-EC) and extended-spectrum beta-lactamase E. coli (ESBL-EC) in children. We collected 1,699 fecal samples from 600 children and 1,871 domestic animal fecal samples from 376 of the same households at up to 5 time points per household over the 3-year study period. We used multivariable log-binomial regression models to estimate relative risks (RR) of 3GCR-EC and ESBL-EC carriage, adjusting for child sex and age, caregiver education, household wealth, and recent child antibiotic use. Risk factors for 3GCR-EC included living within 5 km of more than 5 commercial food animal operations (RR: 1.26; 95% confidence interval (CI): 1.10, 1.45; p-value: 0.001), household pig ownership (RR: 1.23; 95% CI: 1.02, 1.48; p-value: 0.030) and child pet contact (RR: 1.23; 95% CI: 1.09, 1.39; p-value: 0.001). Risk factors for ESBL-EC were dog ownership (RR: 1.35; 95% CI: 1.00, 1.83; p-value: 0.053), child pet contact (RR: 1.54; 95% CI: 1.10, 2.16; p-value: 0.012), and placing animal feces on household land/crops (RR: 1.63; 95% CI: 1.09, 2.46; p-value: 0.019). The primary limitations of this study are the use of proxy and self-reported exposure measures and the use of a single beta-lactamase drug (ceftazidime with clavulanic acid) in combination disk diffusion tests for ESBL confirmation, potentially underestimating phenotypic ESBL production among cephalosporin-resistant E. coli isolates. To improve ESBL determination, it is recommended to use 2 combination disk diffusion tests (ceftazidime with clavulanic acid and cefotaxime with clavulanic acid) for ESBL confirmatory testing. Future studies should also characterize transmission pathways by assessing antibiotic resistance in commercial food animals and environmental reservoirs. CONCLUSIONS: In this study, we observed an increase in enteric colonization of antibiotic-resistant bacteria among children with exposures to domestic animals and their waste in the household environment and children living in areas with a higher density of commercial food animal production operations.

Evolutionary Dynamics of Oropouche Virus in South America.

The Amazon basin is home to numerous arthropod-borne viral pathogens that cause febrile disease in humans. Among these, Oropouche orthobunyavirus (OROV) is a relatively understudied member of the genus Orthobunyavirus, family Peribunyaviridae, that causes periodic outbreaks in human populations in Brazil and other South American countries. Although several studies have described the genetic diversity of the virus, the evolutionary processes that shape the OROV genome remain poorly understood. Here, we present a comprehensive study of the genomic dynamics of OROV that encompasses phylogenetic analysis, evolutionary rate estimates, inference of natural selective pressures, recombination and reassortment, and structural analysis of OROV variants. Our study includes all available published sequences, as well as a set of new OROV genome sequences obtained from patients in Ecuador, representing the first set of genomes from this country. Our results show differing evolutionary processes on the three segments that comprise the viral genome. We infer differing times of the most recent common ancestors of the genome segments and propose that this can be explained by cryptic reassortment. We also present the discovery of previously unobserved putative N-linked glycosylation sites, as well as codons that evolve under positive selection on the viral surface proteins, and discuss the potential role of these features in the evolution of OROV through a combined phylogenetic and structural approach.IMPORTANCE The emergence and reemergence of pathogens such as Zika virus, chikungunya virus, and yellow fever virus have drawn attention toward other cocirculating arboviruses in South America. Oropouche virus (OROV) is a poorly studied pathogen responsible for over a dozen outbreaks since the early 1960s and represents a public health burden to countries such as Brazil, Panama, and Peru. OROV is likely underreported since its symptomatology can be easily confounded with other febrile illnesses (e.g., dengue fever and leptospirosis) and point-of-care testing for the virus is still uncommon. With limited data, there is a need to optimize the information currently available. Analysis of OROV genomes can help us understand how the virus circulates in nature and can reveal the evolutionary forces that shape the genetic diversity of the virus, which has implications for molecular diagnostics and the design of potential vaccines.

Leptospira in river and soil in a highly endemic area of Ecuador.

BACKGROUND: Leptospira are shed into the environment via urine of infected animals. Rivers are thought to be an important risk factor for transmission to humans, though much is unknown about the types of environment or characteristics that favor survival. To address this, we screened for Leptospira DNA in two rivers in rural Ecuador where Leptospirosis is endemic. RESULTS: We collected 112 longitudinal samples and recorded pH, temperature, river depth, precipitation, and dissolved oxygen. We also performed a series of three experiments designed to provide insight into Leptospira presence in the soil. In the first soil experiment, we characterized prevalence and co-occurrence of Leptospira with other bacterial taxa in the soil at dispersed sites along the rivers (n = 64). In the second soil experiment, we collected 24 river samples and 48 soil samples at three points along eight transects to compare the likelihood of finding Leptospira in the river and on the shore at different distances from the river. In a third experiment, we tested whether Leptospira presence is associated with soil moisture by collecting 25 soil samples from two different sites. In our river experiment, we found pathogenic Leptospira in only 4 (3.7%) of samples. In contrast, pathogenic Leptospira species were found in 22% of shore soil at dispersed sites, 16.7% of soil samples (compared to 4.2% of river samples) in the transects, and 40% of soil samples to test for associations with soil moisture. CONCLUSIONS: Our data are limited to two sites in a highly endemic area, but the scarcity of Leptospira DNA in the river is not consistent with the widespread contention of the importance of river water for leptospirosis transmission. While Leptospira may be shed directly into the river, onto the shores, or washed into the river from more remote sites, massive dilution and limited persistence in rivers may reduce the environmental load and therefore, the epidemiological significance of such sources. It is also possible that transmission may occur more frequently on shores where people are liable to be barefoot. Molecular studies that further explore the role of rivers and water bodies in the epidemiology of leptospirosis are needed.

Metagenomic Signatures of Gut Infections Caused by Different Escherichia coli Pathotypes.

Escherichia coli is a leading contributor to infectious diarrhea and child mortality worldwide, but it remains unknown how alterations in the gut microbiome vary for distinct E. coli pathotype infections and whether these signatures can be used for diagnostic purposes. Further, the majority of enteric diarrheal infections are not diagnosed with respect to their etiological agent(s) due to technical challenges. To address these issues, we devised a novel approach that combined traditional, isolate-based and molecular-biology techniques with metagenomics analysis of stool samples and epidemiological data. Application of this pipeline to children enrolled in a case-control study of diarrhea in Ecuador showed that, in about half of the cases where an E. coli pathotype was detected by culture and PCR, E. coli was likely not the causative agent based on the metagenome-derived low relative abundance, the level of clonality, and/or the virulence gene content. Our results also showed that diffuse adherent E. coli (DAEC), a pathotype that is generally underrepresented in previous studies of diarrhea and thus, thought not to be highly virulent, caused several small-scale diarrheal outbreaks across a rural to urban gradient in Ecuador. DAEC infections were uniquely accompanied by coelution of large amounts of human DNA and conferred significant shifts in the gut microbiome composition relative to controls or infections caused by other E. coli pathotypes. Our study shows that diarrheal infections can be efficiently diagnosed for their etiological agent and categorized based on their effects on the gut microbiome using metagenomic tools, which opens new possibilities for diagnostics and treatment.IMPORTANCEE. coli infectious diarrhea is an important contributor to child mortality worldwide. However, diagnosing and thus treating E. coli infections remain challenging due to technical and other reasons associated with the limitations of the traditional culture-based techniques and the requirement to apply Koch's postulates. In this study, we integrated traditional microbiology techniques with metagenomics and epidemiological data in order to identify cases of diarrhea where E. coli was most likely the causative disease agent and evaluate specific signatures in the disease-state gut microbiome that distinguish between diffuse adherent, enterotoxigenic, and enteropathogenic E. coli pathotypes. Therefore, our methodology and results should be highly relevant for diagnosing and treating diarrheal infections and have important applications in public health.

Locals get travellers' diarrhoea too: risk factors for diarrhoeal illness and pathogenic Escherichia coli infection across an urban-rural gradient in Ecuador.

OBJECTIVES: Diarrhoea is a common and well-studied cause of illness afflicting international travellers. However, traveller's diarrhoea can also result from travel between high and low disease transmission regions within a country, which is the focus of this study. METHODS: We recruited participants for a case-control study of diarrhoea at four sites along an urban-rural gradient in Northern Ecuador: Quito, Esmeraldas, Borbón and rural communities outside of Borbón. At each of these sites, approximately 100 subjects with diarrhoea (cases) were recruited from Ministry of Health clinics and were age-matched with subjects visiting the same clinics for other complaints (controls). RESULTS: Travellers to urban destinations had higher risk of diarrhoea and diarrhoeagenic Escherichia coli (DEC) infections. Travel to Quito was associated with diarrhoea (aOR = 2.01, 95% CI = 1.10-3.68) and travel to Guayaquil (another urban centre in Ecuador) was associated with Diffuse Adherent E. coli infection (OR = 2.09, 95% CI = 1.01-4.33). Compared to those not travelling, urban origins were also associated with greater risk of diarrhoea in Esmeraldas (aOR = 2.28, 95% CI = 1.20-4.41), and with higher risk of diarrhoeagenic E. coli infections in Quito (aOR = 2.61, 95% CI = 1.16-5.86), with >50% of travel from Quito and Esmeraldas specified to another urban destination. CONCLUSIONS: This study suggests that individuals travelling from lower-transmission regions (rural areas) to higher transmission regions (urban centres) within a single country are at a greater risk of acquiring a diarrhoea-related illness. Investments to improve water, sanitation and hygiene conditions in urban areas could have impacts on outlying rural areas within a given country.

Isolation of Oropouche Virus from Febrile Patient, Ecuador.

We report identification of an Oropouche virus strain in a febrile patient from Ecuador by using metagenomic sequencing and real-time reverse transcription PCR. Virus was isolated from patient serum by using Vero cells. Phylogenetic analysis of the whole-genome sequence showed the virus to be similar to a strain from Peru.

The Role of Mobile Genetic Elements in the Spread of Antimicrobial-Resistant Escherichia coli From Chickens to Humans in Small-Scale Production Poultry Operations in Rural Ecuador.

Small-scale production poultry operations are increasingly common worldwide. To investigate how these operations influence antimicrobial resistance and mobile genetic elements (MGEs), Escherichia coli isolates were sampled from small-scale production birds (raised in confined spaces with antibiotics in feed), household birds (no movement constraints; fed on scraps), and humans associated with these birds in rural Ecuador (2010-2012). Isolates were screened for genes associated with MGEs as well as phenotypic resistance to 12 antibiotics. Isolates from small-scale production birds had significantly elevated odds of resistance to 7 antibiotics and presence of MGE genes compared with household birds (adjusted odds ratio (OR) range = 2.2-87.9). Isolates from humans associated with small-scale production birds had elevated odds of carrying an integron (adjusted OR = 2.0; 95% confidence interval (CI): 1.06, 3.83) compared with humans associated with household birds, as well as resistance to sulfisoxazole (adjusted OR = 1.9; 95% CI: 1.01, 3.60) and trimethoprim/sulfamethoxazole (adjusted OR = 2.1; 95% CI: 1.13, 3.95). Stratifying by the presence of MGEs revealed antibiotic groups that are explained by biological links to MGEs; in particular, resistance to sulfisoxazole, trimethoprim/sulfamethoxazole, or tetracycline was highest among birds and humans when MGE exposures were present. Small-scale production poultry operations might select for isolates carrying MGEs, contributing to elevated levels of resistance in this setting.

Antibiotic Resistome Associated with Small-Scale Poultry Production in Rural Ecuador.

Small-scale poultry farming is common in rural communities across the developing world. To examine the extent to which small-scale poultry farming serves as a reservoir for resistance determinants, the resistome of fecal samples was compared between production chickens that received antibiotics and free-ranging household chickens that received no antibiotics from a rural village in northern Ecuador. A qPCR array was used to quantify antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) using 248 primer pairs; and the microbiome structure was analyzed via 16S rRNA gene sequencing. A large number of ARGs (148) and MGEs (29) were detected. The ARG richness in production chickens was significantly higher than that of household chickens with an average of 15 more genes detected ( p < 0.01). Moreover, ARGs and MGEs were much more abundant in production chickens than in household chickens (up to a 157-fold difference). Production chicken samples had significantly lower taxonomic diversity and were more abundant in Gammaproteobacteria, Betaproteobacteria, and Flavobacteria. The high abundance and diversity of ARGs and MGEs found in small-scale poultry farming was comparable to the levels previously found in large scale animal production, suggesting that these chickens could act as a local reservoir for spreading ARGs into rural communities.

Detection of Zoonotic Enteropathogens in Children and Domestic Animals in a Semirural Community in Ecuador.

UNLABELLED: Animals are important reservoirs of zoonotic enteropathogens, and transmission to humans occurs more frequently in low- and middle-income countries (LMICs), where small-scale livestock production is common. In this study, we investigated the presence of zoonotic enteropathogens in stool samples from 64 asymptomatic children and 203 domestic animals of 62 households in a semirural community in Ecuador between June and August 2014. Multilocus sequence typing (MLST) was used to assess zoonotic transmission of Campylobacter jejuni and atypical enteropathogenic Escherichia coli (aEPEC), which were the most prevalent bacterial pathogens in children and domestic animals (30.7% and 10.5%, respectively). Four sequence types (STs) of C. jejuni and four STs of aEPEC were identical between children and domestic animals. The apparent sources of human infection were chickens, dogs, guinea pigs, and rabbits for C. jejuni and pigs, dogs, and chickens for aEPEC. Other pathogens detected in children and domestic animals were Giardia lamblia (13.1%), Cryptosporidium parvum (1.1%), and Shiga toxin-producing E. coli (STEC) (2.6%). Salmonella enterica was detected in 5 dogs and Yersinia enterocolitica was identified in 1 pig. Even though we identified 7 enteric pathogens in children, we encountered evidence of active transmission between domestic animals and humans only for C. jejuni and aEPEC. We also found evidence that C. jejuni strains from chickens were more likely to be transmitted to humans than those coming from other domestic animals. Our findings demonstrate the complex nature of enteropathogen transmission between domestic animals and humans and stress the need for further studies. IMPORTANCE: We found evidence that Campylobacter jejuni, Giardia, and aEPEC organisms were the most common zoonotic enteropathogens in children and domestic animals in a region close to Quito, the capital of Ecuador. Genetic analysis of the isolates suggests transmission of some genotypes of C. jejuni and aEPEC from domestic animals to humans in this region. We also found that the genotypes associated with C. jejuni from chickens were present more often in children than were those from other domestic animals. The potential environmental factors associated with transmission of these pathogens to humans then are discussed.

Prevalence of human papillomavirus types in cervical cancerous and precancerous lesions of Ecuadorian women.

Human Papillomavirus (HPV) is the most common sexually transmitted infection worldwide and it is responsible for most cases of uterine cancer. In Ecuador there is limited information about HPV types (and variants) in cancerous lesions; however, identifying the type-specific HPV prevalence in cervical lesions of women living in Ecuador is important to better predict the impact of HPV prophylactic vaccination in this country. We studied the prevalence of HPV types in cervical cancerous or precancerous lesions from 164 Ecuadorian women and found that 86.0% were HPV positive. The most common types were HPV16 (41.8%) and HPV58 (30.5%). Interestingly, HPV18 was detected only in 2.8% of the HPV-positive samples. Fifteen DNA sequences (genes E6 and L1) from 16 samples positive for HPV16 belonged to the European lineage, considered one of the least carcinogenic lineages, and 1 (6.25%) to the Asian-American lineage. Similar analysis in 12 HPV58 positive samples showed that 10 (83.3%) sequences grouped in sublineage A2, which belongs to the oldest HPV58 lineage, 1 belonged to A3 and 1 to lineage C. This study suggests that the currently used HPV vaccines (bivalent and tetravalent) may have lower effectiveness in Ecuador than in other geographic locations where HPV18 is more prevalent.

The Role of the Microbiome in the Relationship of Asthma and Affective Disorders.

The effect of stress, anxiety and other affective states on inflammatory conditions such as asthma is well documented. Although several immune pathway mechanisms have been proposed and studied, they cannot fully explain the relationship. In this chapter we present a new perspective on asthma development and exacerbation that integrates findings on the role of psychological factors in asthma with the microbiome and the hygiene hypothesis in asthma development.

High Prevalence of Intermediate Leptospira spp. DNA in Febrile Humans from Urban and Rural Ecuador.

Leptospira spp., which comprise 3 clusters (pathogenic, saprophytic, and intermediate) that vary in pathogenicity, infect >1 million persons worldwide each year. The disease burden of the intermediate leptospires is unclear. To increase knowledge of this cluster, we used new molecular approaches to characterize Leptospira spp. in 464 samples from febrile patients in rural, semiurban, and urban communities in Ecuador; in 20 samples from nonfebrile persons in the rural community; and in 206 samples from animals in the semiurban community. We observed a higher percentage of leptospiral DNA-positive samples from febrile persons in rural (64%) versus urban (21%) and semiurban (25%) communities; no leptospires were detected in nonfebrile persons. The percentage of intermediate cluster strains in humans (96%) was higher than that of pathogenic cluster strains (4%); strains in animal samples belonged to intermediate (49%) and pathogenic (51%) clusters. Intermediate cluster strains may be causing a substantial amount of fever in coastal Ecuador.

Effects of selection pressure and genetic association on the relationship between antibiotic resistance and virulence in Escherichia coli.

Antibiotic selection pressure and genetic associations may lead to the cooccurrence of resistance and virulence in individual pathogens. However, there is a lack of rigorous epidemiological evidence that demonstrates the cooccurrence of resistance and virulence at the population level. Using samples from a population-based case-control study in 25 villages in rural Ecuador, we characterized resistance to 12 antibiotics among pathogenic (n = 86) and commensal (n = 761) Escherichia coli isolates, classified by the presence or absence of known diarrheagenic virulence factor genes. The prevalences of resistance to single and multiple antibiotics were significantly higher for pathogenic isolates than for commensal isolates. Using a generalized estimating equation, antibiotic resistance was independently associated with virulence factor carriage, case status, and antibiotic use (for these respective factors: odds ratio [OR] = 3.0, with a 95% confidence interval [CI] of 1.7 to 5.1; OR = 2.0, with a 95% CI of 1.3 to 3.0; and OR = 1.5, with a 95% CI of 0.9 to 2.5). Virulence factor carriage was more strongly related to antibiotic resistance than antibiotic use for all antibiotics examined, with the exception of fluoroquinolones, gentamicin, and cefotaxime. This study provides epidemiological evidence that antibiotic resistance and virulence factor carriage are linked in E. coli populations in a community setting. Further, these data suggest that while the cooccurrence of resistance and virulence in E. coli is partially due to antibiotic selection pressure, it is also genetically determined. These findings should be considered in developing strategies for treating infections and controlling for antibiotic resistance.

IS26 drives the dissemination of bla CTX-M genes in an Ecuadorian community.

IMPORTANCE: The horizontal gene transfer events are the major contributors to the current spread of CTX-M-encoding genes, the most common extended-spectrum ß-lactamase (ESBL), and many clinically crucial antimicrobial resistance (AMR) genes. This study presents evidence of the critical role of IS26 transposable element for the mobility of bla CTX-M gene among Escherichia coli isolates from children and domestic animals in the community. We suggest that the nucleotide sequences of IS26-bla CTX-M could be used to study bla CTX-M transmission between humans, domestic animals, and the environment, because understanding of the dissemination patterns of AMR genes is critical to implement effective measures to slow down the dissemination of these clinically important genes.

Presence of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Food-Producing and Companion Animals and Wildlife on Small-Holder Farms of Floreana Island, Galápagos Islands.

Background: Antimicrobial resistance (AR) has led to increasing human and animal morbidity and mortality and negative consequences for the environment. AR among Escherichia coli (EC) is on the rise, with serious concerns about extended-spectrum ß-lactamase-producing E. coli (ESBL-EC). In the Galápagos Islands, where antimicrobials are available without a prescription, growing demands for food production can drive antimicrobial use. Food producing animals are at the interface of wildlife and environmental health on the smallest human-inhabited Galápagos Island, Floreana. We sought to determine if ESBL-EC were present in Floreana Island farm animal species and nearby wildlife and the relatedness of ESBL-EC isolates identified. Materials and Methods: During July 4-5, 2022, we visited 8 multispecies farms, representing 75% of food-producing animal production on Floreana, and collected 227 fecal samples from farm animals and wildlife. Each sample was plated on MacConkey agar supplemented with cefotaxime (4 µg/mL). Results: ESBL-EC was isolated from 20 (9%) fecal samples collected from pigs (N = 10), chickens (N = 6), wildlife (N = 3), and dog (N = 1). All ESBL-EC isolates were from samples taken at three (38%) of the eight farms. Fifteen (75%) of the ESBL-EC isolates were from a single farm. All ESBL-EC isolates were multidrug resistant. The most prevalent ESBL genes belonged to the blaCTX-M group. Among the typeable isolates from the farm with the largest proportion of ESBL-EC isolates (N = 14), we observed nine unique pulsed-field gel electrophoresis (PFGE) patterns, with identical patterns present across pig and chicken isolates. PFGE patterns in the three farms with ESBL-EC isolates were different. Conclusions: These results lend support for future routine AR monitoring activities at the livestock-wildlife interface in Galápagos to characterize potential interspecies transmission of AR bacteria and AR genes in this unique protected ecosystem, and the related human, animal, and environmental health impacts, and to formulate interventions to reduce AR spread in this setting.

Endogenous origin of Pseudomonas aeruginosa infecting hospitalized patients in Ecuador.

Recent evidence suggests that Pseudomonas aeruginosa, a bacterium that has the ability to cause deadly infections in hospitalized patients, could originate in the patient's own flora. We employed the Oxford Nanopore platform to obtain whole genome sequences (WGS) from clinical and rectal screen P. aeruginosa strains belonging to 15 patients from two hospitals. Our study found evidence that clinical and rectal isolates were clonal, with some evidence suggesting that the infecting strain was present in the patient's intestine at the time of admission, ruling out hospital acquisition. The use of WGS analysis is crucial to detect alternative sources of P. aeruginosa to develop new preventive measures against these serious infections.

Transition in the cause of fever from malaria to dengue, Northwestern Ecuador, 1990-2011.

In tropical areas, the predominant cause of fever has historically been malaria. However by 2011, among febrile patients in northwestern Ecuador, dengue was identified in 42% and malaria in none. This finding suggests a transition in the cause of fever from malaria to other illnesses, such as dengue.

Treatment of acid rock drainage using a sulphate-reducing bioreactor with a limestone precolumn.

Sulphate reducing bacteria (SRB) offer promise for the treatment of mine waste due to their effectiveness removing toxic heavy metals as highly insoluble metal sulphides and their ability to generate alkalinity. The main objective of this study was to develop a treatment composed of a sulphate-reducing bioreactor with a limestone precolumn for the removal of Cu(II) from a synthetic ARD. The purpose of the limestone column was to increase the pH values and decrease the level of Cu in the effluent to prevent SRB inhibition. The system was fed with a pH-2.7 synthetic ARD containing Cu(II) (10-40 mg/L), sulphate (2000 mg/L) and acetate (2.5 g COD/L) for 150 days. Copper removal efficiencies in the two-stage system were very high (95-99%), with a final concentration of 0.53 mg/L Cu, and almost complete removal occurred in the limestone precolumn. In the same manner, the acidity of the synthetic ARD was effectively reduced in the limestone precolumn to 7.3 and the pH was raised in the bioreactor (7.3-8.0). COD consumption by methanogens was predominant from day 0-118, but SRB dominated at the end of the experiment (day 150) when the average COD removal and sulphide production were 74.8% and 61.7%, respectively. Study of the microbial taxonomic composition in the bioreactor revealed that Methanosarcina and Methanosaeta were the most prevalent methanogens while the genera Desulfotomaculum and Syntrophobacter were the dominant SRB. Among the SRB identified Desulfotomaculum intricatum (99% identity) and Desulfotomaculum acetoxidans (96%) were the most abundant sequences of bacteria capable of using acetate.