Liseth Salinas

Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-producing E. coli ) pose a growing global health threat. Although Latin America has been identified as a global hotspot of antimicrobial resistance, the zoonotic contribution to drug-resistant infections in the region remains poorly defined. We analyzed 137 clinical ESBL-producing E. coli isolates from urinary tract infections (UTIs) in Quito, Ecuador, applying a Bayesian latent class model informed by host-associated mobile genetic elements to estimate the fraction of infections attributable to food-animal sources. We estimated that 25.5% (35/137) of UTI isolates were putative zoonotic cases. This proportion rose to 42.5% after excluding ST131- H 30, a human-associated pandemic lineage. Putative zoonotic isolates were enriched for animal-associated β-lactamase genes (e.g., bla _TEM-1B , bla _CTX-M-65 ), lacked human-associated markers such as bla _OXA-1 , and exhibited diverse antimicrobial resistance gene profiles resembling those observed among food-animal isolates. These isolates were also enriched for ColV-associated virulence genes typically linked to avian pathogenic E. coli . Putative zoonotic strains contributed substantially to third-generation cephalosporin-resistant UTIs in Quito, Ecuador, challenging assumptions derived from high-income settings that such infections are driven predominantly by human-to-human transmission. These findings highlight the importance of integrated One Health surveillance and mitigation, particularly in low- and middle-income countries where gaps in water, sanitation, and hygiene (WASH) may interact with antimicrobial use in food production to amplify antimicrobial resistance transmission. IMPORTANCE: ESBL-producing E. coli have rapidly emerged as a major global antimicrobial resistance threat. In Latin America, cephalosporins are commonly used in food-animal production, fueling the emergence of ESBL-producing E. coli . In low- and middle-income countries, excessive antimicrobial use driven by poorly regulated over-the-counter sales, combined with inadequate water, sanitation, and hygiene (WASH) infrastructure, can facilitate antimicrobial-resistant pathogen transmission from food animals to humans. Using a novel statistical-genomic approach, we found that over one in four cephalosporin-resistant UTIs in Quito, Ecuador, may be caused by E. coli strains originating from food animals. Our findings highlight the public health risks associated with antimicrobial use in food-animal production and the role of environmental and infrastructure-related vulnerabilities. As global demand for animal protein continues rising in middle-income countries, controlling zoonotic antimicrobial resistance transmission becomes increasingly urgent for protecting human health through integrated One Health strategies.

Background/Objectives: Integrons are genetic platforms that allow bacteria to acquire antimicrobial resistance (AMR) genes, making them a focal point for many AMR studies and surveillance programs. This study investigated how the prevalence of integrons (intI and attI genes) in third-generation cephalosporin-resistant E. coli (3GCR-Ec) varied across three different sources (i.e., healthy children, domestic animals and urinary tract infections). The study aimed to determine how different classes of AMR genes vary among 3GCR-Ec with integrons present versus those where integrons are absent. Methods: We analyzed 3GCR-Ec isolates collected from semirural parishes of Eastern Quito, Ecuador, that included: (1) 3GCR-Ec from healthy children (n = 946), (2) 3GCR-Ec from domestic animal species (n = 673), and 3GCR-Ec from patients with urinary tract infections (UTIs) (n = 138). Genomic analyses were performed for all 1757 sequences to determine how the presence and absence of integrons was associated with AMR gene carriage. Results: Among the total sequences of 3GCR-Ec evaluated across all datasets, nearly one-third (31%) were integron-negative. 3GCR-Ec from UTI patients, however, had a higher percentage containing integrons (79%). Across all sets of 3GCR-EC, integron-positive isolates carried an average of 10.3 (±3.0 SD) AMR genes versus 4.8 (±2.5 SD) AMR genes in integron-negative isolates. This study found that between 21% to 33% of 3GCR-Ec across the three different sources lacked integrons but maintained the ability to carry diverse classes of AMR genes, including beta-lactams, aminoglycosides, tetracyclines, and multidrug resistance mechanisms (e.g., general-purpose efflux pumps). Conclusions: While integrons were associated with greater AMR genes on average, the study highlights that solely relying on integrons for tracking drug-resistant bacteria misses a substantive portion of AMR that is present in integron-negative strains.

Antimicrobial resistance (AMR) is a major threat to global public health that continues to grow owing to selective pressure caused by the use and overuse of antimicrobial drugs. Resistance spread by plasmids is of special concern, as they can mediate a wide distribution of AMR genes, including those encoding extended-spectrum β-lactamases (ESBLs). The CTX-M family of ESBLs has rapidly spread worldwide, playing a large role in the declining effectiveness of third-generation cephalosporins. This rapid spread across the planet is puzzling given that plasmids carrying AMR genes have been hypothesized to incur a fitness cost to their hosts in the absence of antibiotics. Here, we focus on a WT plasmid that carries the bla _{CTX-M 55} ESBL gene. We examine its conjugation rates and use head-to-head competitions to assay its associated fitness costs in both laboratory and wild Escherichia coli strains. We found that the wild strains exhibit intermediate conjugation levels, falling between two high-conjugation and two low-conjugation laboratory strains, the latter being older and more ancestral. We also show that the plasmid increases the fitness of both WT and lab strains when grown in lysogeny broth and Davis–Mingioli media without antibiotics, which might stem from metabolic benefits conferred on the host, or from interactions between the host and the rifampicin-resistant mutation we used as a selective marker. Laboratory strains displayed higher conjugation frequencies compared to WT strains. The exception was a low-passage K-12 strain, suggesting that prolonged laboratory cultivation may have compromised bacterial defences against plasmids. Despite low transfer rates among WT E. coli, the plasmid carried low fitness cost in minimal medium but conferred improved fitness in enriched medium, indicating a complex interplay between plasmids, host genetics and environmental conditions. Our findings reveal an intricate relationship between plasmid carriage and bacterial fitness. Moreover, they show that resistance plasmids can confer adaptive advantages to their hosts beyond AMR. Altogether, these results highlight that a closer study of plasmid dynamics is critical for developing a secure understanding of how they evolve and affect bacterial adaptability that is necessary for combating resistance spread.

To investigate the nucleotide sequences associated with transposable elements carrying bla_CTX-M allelic variants as potential markers for the transmission of antimicrobial resistance genes between domestic animals, humans and the environment. We conducted whole-genome sequencing and analyzed the nucleotide sequences of most abundant bla_CTX-M allelic variants (bla_CTX-M-27, bla_CTX-M-55, and bla_CTX-M-65) in commensal Escherichia coli (n = 20) from household members in Quito and uropathogenic E. coli (UPEC) (n = 149) isolated from nine clinics in Quito, Ecuador. The Ecuadorian commensal E. coli and UPEC displayed identical nucleotide sequences surrounding the bla_CTX-M gene and the synteny was similar to those found in other parts of the world; however phylogenetic analysis indicated that the genetic environments in Ecuadorian isolates were unique. These findings suggest that the nucleotide sequences flanking the bla_CTX-M genes may be useful for resolving ARG transmission pathways, especially inter-regional analyses.

Antibiotic resistance (ABR) is a critical and growing global challenge, especially in low‐ and middle‐income countries. Ecuador has made great progress in connecting households to piped water supplies; however, the collection and treatment of domestic wastewater has lagged. This infrastructural gap may be accelerating the spread of ABR into surface waters used downstream for irrigation. We studied the contributions of a small town in Ecuador to the prevalence of extended‐spectrum β‐lactamase‐producing Escherichia coli in a glacial stream used for irrigating crops. The study analyzed water samples upstream (n = 60) and downstream (n = 60) of the town of Píntag as well as 30 lettuce samples irrigated by surface waters downstream of the town. A subset of third generation cephalosporin resistant E. coli (3GCR‐EC) isolates (n = 58) were sequenced to characterize antibiotic resistance genes and pathogenic lineages. Our results showed that there was nearly a three‐log increase in mean E. coli colony forming units in the downstream samples versus upstream. At the upstream sites above the town of Píntag, 6.7% of water samples were positive for 3GCR‐EC compared to 100% of samples collected at the downstream sites. Additionally, 70.1% of sequenced 3GCR‐EC isolates collected at downstream sites carried bla_CTX‐M genes and 3.4% belonged to pandemic lineages ST131 and ST10. As countries develop household piped water infrastructure, attention should focus on how the lack of domestic wastewater collection and treatment may accelerate the spread of ABR in waterways and the food system.

There is little information available on antibiotic resistance (ABR) within shrimp aquaculture environments. The aim of this study was to investigate the presence of antibiotic resistance genes (ARGs) in shrimp farming operations in Atacames, Ecuador. Water samples (n = 162) and shrimp samples (n = 54) were collected from three shrimp farming operations. Samples were cultured and a subset of isolates that grew in the presence of ceftriaxone, a third-generation cephalosporin, were analyzed using whole-genome sequencing (WGS). Among the sequenced isolates (n = 44), 73% of the isolates contained at least one ARG and the average number of ARGs per isolate was two, with a median of 3.5 ARGs. Antibiotic resistance genes that confer resistance to the β-lactam class of antibiotics were observed in 65% of the sequenced isolates from water (20/31) and 54% of the isolates from shrimp (7/13). We identified 61 different ARGs across the 44 sequenced isolates, which conferred resistance to nine antibiotic classes. Over half of all sequenced isolates (59%, n = 26) carried ARGs that confer resistance to more than one class of antibiotics. ARGs for certain antibiotic classes were more common, including beta-lactams (26 ARGs); aminoglycosides (11 ARGs); chloramphenicol (three ARGs); and trimethoprim (four ARGs). Sequenced isolates consisted of a diverse array of bacterial orders and species, including Escherichia coli (48%), Klebsiella pneumoniae (7%), Aeromonadales (7%), Pseudomonadales (16%), Enterobacter cloacae (2%), and Citrobacter freundii (2%). Many ARGs were shared across diverse species, underscoring the risk of horizontal gene transfer in these environments. This study indicated the widespread presence of extended-spectrum β-lactamase (ESBL) genes in shrimp aquaculture, including bla_CTX-M, bla_SHV, and bla_TEM genes. Increased antibiotic resistance surveillance of shrimp farms and identification of aquaculture operation-level risk factors, such as antibiotic use, will likely be important for mitigating the spread of ARGs of clinical significance.

The rapid dissemination of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales, mainly Escherichia coli carrying bla _CTX-M genes, is a major public health concern due to its successful spread in hospital settings as well as among humans and animals in the community. We characterized ESBL-producing E. coli isolates from children and domestic animals in semirural communities of Ecuador to assess the contribution of horizontal gene transfer of the bla _CTX-M genes among E. coli isolates. From 20 selected E. coli isolates (from children and domestic animals) harboring bla _CTX-M allelic variants, we identified 16 plasmids carrying bla _CTX-M-55 ( n = 9), bla _CTX-M-65 ( n = 5), and bla _CTX-M-27 ( n = 2), as well as four chromosomes carrying bla _CTX-M-65 . The backbone structure of plasmids, including replication, maintenance, and plasmid transfer genes, and the synteny was conserved in all plasmids carrying the same bla _CTX-M allelic variant. In all plasmids and chromosomes, the bla _CTX-M genes were bracketed by two IS 26 transposable elements. This study highlights the critical role of the IS 26 transposable element for the current mobility of bla _CTX-M genes among plasmids or from plasmids to chromosomes, suggesting that IS 26-bla _CTX-M brackets could be used to study bla _CTX-M transmission between humans, domestic animals, and the environment. IMPORTANCE: The horizontal gene transfer events are the major contributors to the current spread of CTX-M-encoding genes, the most common extended-spectrum β-lactamase (ESBL), and many clinically crucial antimicrobial resistance (AMR) genes. This study presents evidence of the critical role of IS 26 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 IS 26 - 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.

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.

The coronavirus 2019 (COVID-19) pandemic has had significant impacts on health systems, population dynamics, public health awareness, and antibiotic stewardship, which could affect antibiotic resistant bacteria (ARB) emergence and transmission. In this study, we aimed to compare knowledge, attitudes, and practices (KAP) of antibiotic use and ARB carriage in Ecuadorian communities before versus after the COVID-19 pandemic began. We leveraged data collected for a repeated measures observational study of third-generation cephalosporin-resistant E. coli (3GCR-EC) carriage among children in semi-rural communities in Quito, Ecuador between July 2018 and September 2021. We included 241 households that participated in surveys and child stool sample collection in 2019, before the pandemic, and in 2021, after the pandemic began. We estimated adjusted Prevalence Ratios (aPR) and 95% Confidence Intervals (CI) using logistic and Poisson regression models. Child antibiotic use in the last 3 months declined from 17% pre-pandemic to 5% in 2021 (aPR: 0.30; 95% CI 0.15, 0.61) and 3GCR-EC carriage among children declined from 40 to 23% (aPR: 0.48; 95% CI 0.32, 0.73). Multi-drug resistance declined from 86 to 70% (aPR: 0.32; 95% CI 0.13; 0.79), the average number of antibiotic resistance genes (ARGs) per 3GCR-EC isolate declined from 9.9 to 7.8 (aPR of 0.79; 95% CI 0.65, 0.96), and the diversity of ARGs was lower in 2021. In the context of Ecuador, where COVID-19 prevention and control measures were strictly enforced after its major cities experienced some of the world’s the highest mortality rates from SARS-CoV-2 infections, antibiotic use and ARB carriage declined in semi-rural communities of Quito from 2019 to 2021.

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 domestic animals, backyard food animals, and commercial food animal production in Ecuadorian communities. Methods & Findings: We conducted a repeated-measures study from 2018-2021 in 7 semi-rural 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 and domestic animals. We used multivariable log-binomial regression models to estimate relative risks (RR) of 3GCR-EC and ESBL-EC carriage. We collected 1,699 child fecal samples from 600 households and 1,871 animal fecal samples from 376 of the same households. Risk factors for 3GCR-EC included living within 5 km of more than 5 commercial food animal operations (RR: 1.36; 95% Confidence Interval: 1.16, 1.59), household pig ownership (1.23; 1.02, 1.48), child pet contact (1.23; 1.09, 1.39), and rarely/never washing hands after contact with animals (1.15; 0.98, 1.34). Risk factors for ESBL-EC were dog ownership (1.43; 1.00, 2.04), child pet contact (1.54; 1.10, 2.16), placing animal feces on household land/crops (1.63; 1.09, 2.46), and combined exposures to both household food animals and commercial food animal operation drainage paths (1.80; 0.94, 3.45). Conclusions: Policies and interventions that improve the safety of animal waste management in communities and in commercial food animal production operations may be necessary to curb the spread of resistant bacteria.