Extended-spectrum β-lactamases genes in Gram-negative isolates from an urban river in Nicaragua

Genes de β-lactamases de espectro estendido em isolados Gram-negativos de um rio urbano na Nicarágua

Abstract

Limited resources and inadequate surveillance systems in developing countries have hindered research on antibiotic resistance gene transfer in aquatic environments. In this context, our study aimed to identify extended-spectrum beta-lactamase gene variants in Gram-negative isolates from the Tipitapa River—a significant Central America ecosystem. Samples were collected and assessed for key water parameters: dissolved oxygen, electrical conductivity, pH, and temperature. We employed a mix of microbiological, biochemical and molecular techniques, including multiplex PCR and sequencing, to characterize bacteria and determine the bla gene variants. Water quality parameters indicated areas impacted by human activities with high mineralization and eutrophication conditions. Among the bacteria analyzed, 48% belonged to the Enterobacteriaceae family, and a significant 88% displayed extended-spectrum beta-lactamases. Sequencing revealed four distinct bla gene variants in 84% of the isolates: bla-SHV-24, bla-SHV-13, bla-TEM-1, and bla-TEM-116, with bla-SHV-24 being the most common (47.62%). This is the first report of bla gene variants in the Tipitapa River, revealing their presence in globally concerning bacteria. The robust methodology enhances surveillance, enables geographic profiling of bla gene variants, and improves our understanding of biochemical patterns and gene transmission dynamics, providing essential insights into the global distribution of antibiotic resistance genes in Central America's natural waters.

Keywords:
antimicrobial resistance; ecosystems functioning; water quality; microbial ecology; environmental microbiology

Resumo

Recursos limitados e sistemas de vigilância inadequados em países em desenvolvimento têm dificultado a pesquisa sobre a transferência de genes de resistência a antibióticos em ambientes aquáticos. Neste contexto, nosso estudo teve como objetivo identificar variantes do gene β-lactamase de espectro estendido em isolados Gram-negativos do Rio Tipitapa, que integra um importante ecossistema da América Central. Foram coletadas amostras e analisados os principais parâmetros da água: oxigênio dissolvido, condutividade elétrica, pH e temperatura. Empregamos uma mistura de técnicas microbiológicas, bioquímicas e moleculares, incluindo PCR multiplex e sequenciamento, para caracterizar bactérias e determinar as variantes do gene bla. Os parâmetros de qualidade da água indicaram áreas impactadas por atividades humanas com alta mineralização e condições de eutrofização. Dentre as bactérias analisadas, 48% pertenciam à família Enterobacteriaceae, e um significativo percentual de 88% apresentava β-lactamase de espectro estendido. O sequenciamento revelou quatro variantes distintas do gene bla em 84% dos isolados: bla-SHV-24, bla-SHV-13, bla-TEM-1 e bla-TEM-116, sendo bla-SHV-24 a mais comum (47,62%). Este é o primeiro relato de variantes do gene bla no Rio Tipitapa, revelando sua presença em bactérias globalmente preocupantes. A metodologia robusta aprimora a vigilância, permite o perfil geográfico das variantes do gene bla e melhora nossa compreensão dos padrões bioquímicos e da dinâmica da transmissão gênica, fornecendo perspectivas essenciais sobre a distribuição global de genes de resistência a antibióticos nas águas naturais da América Central.

Palavras-chave:
resistência antimicrobiana; funcionamento de ecossistemas; qualidade da água; ecologia microbiana; microbiologia ambiental

1. Introduction

Antibiotic resistance is currently a major health concern worldwide (Aslam et al., 2021; Munk et al., 2022). It is estimated that by 2050, 10 million human deaths annually will be associated with antimicrobial resistance (AMR). In 2019, out of the 1.2 million deaths directly associated with resistant bacterial infections worldwide, 89,100 occurred in Latin America, with Central Latin America being the region with the highest number of deaths (28,300) attributable to AMR (Murray et al., 2022).

Gram-negative bacteria present resistance profiles mediated by beta-lactamases, one of the major groups of enzymes that resist the antibiotic effect by hydrolyzing the amide bond of the β-lactam ring. Extended-spectrum β-lactamases (ESBL) in Gram-negative bacteria confer the ability to hydrolyze third-generation antibiotics, namely cephalosporins, and aztreonam, widely prescribed as broad-spectrum antibiotics in humans (Collignon et al., 2016).

ESBL-encoding genes, such as bla-SHV, bla-TEM, bla-CTX-M, and bla-OXA (Abrar et al., 2019), are prevalent and frequently associated with mobile genetic elements like plasmids, transposons, or insertion sequences, which promote the dissemination of resistance genes (Partridge et al., 2018). Due to the tendency of bacteria to develop and transfer mutations in these genes, more than 800 gene variants collectively have been reported (Rawat and Nair, 2010). Molecular diagnostic methods allow the identification of bla gene variants (Delgado et al., 2016).

Numerous studies have demonstrated that antibiotic resistance can be transferred among humans, animals, and the environment, with water playing a particularly significant role (Alcalá et al., 2016). Aquatic ecosystems have been described as excellent environments for the interaction and transfer of antibiotic resistance genes between bacteria (Hooban et al., 2020). This transfer occurs because genetic variants possess specific biochemical activities and distinct patterns of resistance, ultimately posing a serious threat to public health. Factors such as water quality, mobile genetic elements, and anthropogenic activities can influence bacterial genetic profiles (Zhou et al., 2017).

Inappropriate use of antibiotics in both humans and animals is a global driver of antibiotic resistance that impacts selective pressure (Irfan et al., 2022). Furthermore, poor sewage disposal and inefficient wastewater treatment can lead to the contamination of water bodies. Low- and Middle-income countries, like those in Central America, face additional challenges with limited access to efficient wastewater treatment plants and inadequate policies and regulations (Domínguez et al., 2021). The prevalence and sources of antibiotic resistance in river-lake systems, especially in less developed countries with limited resources and understudied ecosystems, are less well-documented compared to other aquatic environments (Chen et al., 2020).

To address these challenges, this study aimed to detect and identify ESBL-producing Gram-negative bacteria in the Tipitapa River—an aquatic ecosystem of significant economic and ecological value in Nicaragua. We employed a combination of microbiological and biochemical methods for bacterial characterization, along with molecular techniques such as multiplex PCR and Sanger sequencing to establish the bla gene variants. Our findings contribute valuable insights to the global understanding of the distribution and variation of antibiotic resistance genes in natural waters of Central America.

2. Materials and Methods

2.1. Study site and sampling

Sampling was conducted in March 2022 during the dry season, encompassing the upper, middle, and low reaches of the Tipitapa River. To ensure a representative selection of sites, proximity to potential sources of pollution was considered as a criterion.

The Tipitapa River is exposed to various anthropogenic impacts, including domestic, agricultural, fishing, and, most notably, hospital and wastewater treatment plant (WWTP) contamination.

Anthropogenic activities and potential sources of pollution were meticulously documented for each sampling site to facilitate further analysis. The sampling points included locations near the mouth of Lake Xolotlán, the municipal hospital's wastewater site, and the farming area. Additionally, samples were collected in the Tisma lagoon, a Ramsar wetland known for its importance in conserving flora, fauna, and migratory birds (Figure 1).

Figure 1
Geographical Location of Sampling Sites. The figure depicts the Tipitapa River basin and the sampling sites situated along the Tipitapa River in central-eastern Nicaragua.

Samples were collected in triplicate using sterile polystyrene bottles, adhering to ISO 19458 (ISO, 2007) standards, and were refrigerated during transport to the laboratory. Within 24 hours of arrival, microbiological analyses were performed on the samples.

During the sample collection, we measured dissolved oxygen (DO) using the FisherBrand Traceable Dissolved Oxygen device. The parameters of electrical conductivity (EC), pH, and temperature were measured with a Milwaukee MW804 pocket tester. To determine total dissolved solids (TDS), we utilized the linear relation with EC (Taylor et al., 2018), defined as (Equation 1):

$$TDS\left(mg/l\right)=0.64\times EC\left(\mu S/cm\right)$$ (1)

2.2. Isolation of bacteria

The methodology described by Amaya et al. (2012) was followed to identify and isolate Gram-negative bacteria. We filtered 250 ml of the collected water samples using sterile membrane filters with a pore size of 0.45 µm (Millipore Corporation, Bedford, MA, USA). Each filter was then deposited on the surface of agar plates used as growth control media. ESBL Agar Brilliance (Oxoid) served as chromogenic selective agar, and MacConkey agar as differential agar. After incubating bacterial colonies at 37 °C for 24 hours, morphologically distinct colonies were collected in triplicate and subcultured to ensure bacterial isolation.

For identification at the species level, we performed biochemical reactions using the API test, following the manufacturer's specifications. The results were analyzed using API 20 E software (Biomérieux, 2010). To preserve each isolate colony, we stored them in 10% skim milk at -80 °C.

2.3. Testing for the ESBL production

Phenotypic characterization of ESBL-producing Gram-negative strains was performed using the double-disk synergy test (DDST) and the Clinical and Laboratory Standards Institute confirmatory test (CLSI, 2022).

In the DDST, we placed amoxicillin beta-lactam/clavulanic acid (AMC) (30 µg/disc) against third-generation cephalosporin antibiotics (30 µg/disc), including ceftriaxone (CRO), ceftazidime (CAZ), or cefotaxime (CTX). The discs were positioned equally spaced apart, and the isolates were incubated at 37 °C for 18 hours. Detection of synergistic effects within the zone where the antibiotics were placed simultaneously demonstrated the enzymatic action of the bacteria, indicating ESBL production (Ejaz et al., 2013).

For the CLSI confirmatory test, we used ceftazidime (CAZ 30 µg) and cefotaxime (CTX 30 µg) alone, as well as combined discs of cefotaxime with amoxicillin beta-lactam/clavulanic acid (CAZ/AMC 30/10 µg) and cefotaxime amoxicillin beta-lactam/clavulanic acid (CTX/AMC 30/10 µg). These discs were placed at a distance of 20 mm from each other and then incubated for 24 hours at 37 °C. A positive result in the CLSI confirmatory test was indicated when the inhibition zone produced by the combined effect of the antibiotics and amoxicillin beta-lactam/clavulanic acid increased greater than 5 mm in comparison to ceftazidime or cefotaxime without amoxicillin beta-lactam/clavulanic acid.

The susceptibility test data were recorded in the Whonet 2021 program. The American Type Culture Collection (ATCC) strains were used as quality control in both DDST and CLSI tests. Klebsiella pneumoniae ATCC 700603 served as a positive control, while Escherichia coli ATCC 25922 was used as a negative control.

2.4. PCR and Sanger sequencing

Identification of each bacterial species was confirmed by Sanger sequencing. DNA extraction was performed using the DNeasy Blood & Tissue Kit (Qiagen, CA, USA), following the manufacturer's instructions. A 1506-bp fragment of the ribosomal 16S Polymerase Chain Reaction (PCR) was amplified using the universal primers fD2 and rD1 (Weisburg et al., 1991).

For the PCR, we used a 12.5 µL reaction volume containing 2 µL of DNA template, 3 µL of deionized H₂O, 6.25 µL of Phusion® High-Fidelity PCR Master Mix with hydrofluoric acid (HF) buffer (New England Biolabs, UK), and 2 μl of each PCR primer (10 µM). The cycling protocol included an initial denaturation step at 98 °C for 45 s; 30 amplification cycles at 98 °C for 5 sec, 56 °C for 30 sec, and 72 °C for 1 min; and a final extension step at 72 °C for 10 min. We then checked the PCR products by electrophoresis in 1.2% agarose with an ethidium bromide stain and purified them using the Exo CIP A and Exo CIP B enzymes (New England Biolabs, USA).

Clean DNA was used for cycle sequencing with the forward primer fD1 (3.2 µM) and BigDye v3.1 (Thermofisher, MA, USA). The sequencing was performed using a SeqStudio genetic analyzer (SeqStudio™, Thermofisher, MA, USA). The obtained sequences were examined using Seq A (Geospiza, Inc., 2024) and blasted in the National Center for Biotechnology Information (NCBI, 2024). Species-level identification was based on >95% percent identity.

2.5. Genotypic characterization of resistant strains by multiplex PCR

For the simultaneous detection of the resistance genes bla-SHV, bla-TEM, bla-CTX-M and bla-OXA, we utilized the universal primers (Fang et al., 2008) and the Multiplex PCR assay (Qiagen, CA, USA). The PCR conditions were the same as described above, with the annealing time extended to 3 minutes at 57 °C. To improve the sensitivity and specificity of gene detection, a nested PCR assay was employed (Carr et al., 2009).

Following the screening of genes obtained through PCR Multiplex, conventional PCR assays were implemented using the same primers mentioned earlier. The cycling protocol included an initial denaturation step at 98 °C for 45 seconds; 30 amplification cycles at 98 °C for 5 seconds, 57 °C for 30 seconds, and 72 °C for 1 minute; and a final extension step at 72 °C for 10 minutes. PCR products were purified using Exo CIP A and Exo CIP B enzymes. For the cycle sequence reaction with BigDye v3.1, forward primers of each amplified gene were used. Sequencing was performed using a SeqStudio genetic analyzer (SeqStudio™, Thermofisher).

2.6. Analysis of sequences bla genes

Following capillary electrophoresis, sequence quality was evaluated and analyzed using Sequencing Analysis Software 6 (Thermofisher, USA). Deduced amino acids were obtained using the software MEGA X and then validated in EMBL (Madeira et al., 2019). Nucleotide and amino acid sequences were compared with those listed in NCBI to identify the types of beta-lactamases.

3. Results

The physicochemical properties of the water were measured at seven different sampling sites along the river (Table 1). The pH value ranged between 7.63 and 8.91. The highest EC and TDS (1.75 mS/cm and 1120 ppm, respectively) were found at the mouth of Lake Xolotlán. The lowest value of OD (2.5 mg/L) was found near the Municipal Hospital at site 2. The temperature at the sampling sites ranged from 29 °C to 32 °C.

Table 1
Physicochemical Characteristics of Water Samples Collected from the Tipitapa River, Nicaragua.

Sampling site	pH	Conductivity (mS/cm)	Dissolved oxygen (mg/L)	Total dissolved solids (ppm)
1. Mouth of Lake Xolotlán	8.94	1.75	5.70	1120
2. Municipal hospital	7.7	1.19	2.50	761
3. Wastewater Treatment Plant (WWTP)	7.63	0.70	3.70	448
4. Farming area	7.94	0.87	5.0	557
5. Tisma lagoon	8.58	0.76	6.0	486
6. Human settelements	8.91	0.27	6.6	173
7. Mouth of Tipitapa River	8.74	0.26	5.2	166
Average	8.35	0.83	5.5	530
Standard deviation	0.57	0.52	0.71	334

Our sampling at seven different sites along the Tipitapa River generated a total of 41 bacterial colonies, out of which 25 colonies (61%) were Gram-negative. Through Sanger sequencing, we identified 12 bacterial colonies (48%) belonging to the Enterobacteriaceae family and 13 (52%) classified as Non-fermenting bacteria. Among the Enterobacteriaceae family, the most frequently identified were Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae (3 isolates each, 12%), followed by Enterobacter bugandensis and Klebsiella variicola (1 isolate of each, 2%).

As for the Non-fermenting bacteria, Aeromonas veronii was the most prevalent (n=6, 24%), followed by Aeromonas hydrophila and Acinetobacter baumannii (2 isolates of each, 8%), and Vibrio cholerae, Providencia rettgeri, Acinetobacter soli, and Aeromonas schubertii (1 isolate of each, 4%).

All Gram-negative isolates were subjected to ESBL production tests using the DDST and CLSI confirmatory tests, as outlined in the Clinical and Laboratory Standards Institute (CLSI, 2022) guidelines (Figure 2). Among the tested bacterial colonies, only four (16%) presented synergy effects, while 23 (92%) showed an increase of more than 5 mm in the diameter of the zone of inhibition. Two colonies (8%) showed a negative ESBL phenotype and did not exhibit an increase of >5 mm in the diameter zone.

Figure 2
ESBL Production Test. (A) Double-disk synergy test (DDST) demonstrating the synergy effect by placing amoxicillin/clavulanic acid (AMC, 30 µg) against third-generation cephalosporin antibiotics (30 µg), such as ceftriaxone (CRO) and cefotaxime (CTX); (B) ESBL production is indicated by an increase in the zone diameter of more than 5 mm in the presence of amoxicillin/clavulanic acid.

The presence of bla genes was examined using Multiplex PCR assays and subsequently confirmed through conventional PCR (Figure 3). Genes mediating ESBL production were detected in 21 (84%) Gram-negative bacterial isolates. The predominant genes were bla-SHV (80.95%) and bla-TEM (42.85%). Notably, 5 (23.80%) of the isolates carried both genes. The bacterial colonies carrying variants of bla-SHV and bla-TEM included Aeromonas (n=6), Acinetobacter (n=3), Enterobacter (n=4), Klebsiella (n=4), Escherichia (n=3), Providencia (n=1) and Vibrio (n=1).

Figure 3
Detection of bla Genes Through PCR Analysis of Gram-Negative Bacterial Isolates. (A) Multiplex PCR assay for the simultaneous detection of bla genes: E. coli (Escherichia coli ATCC 35218 - bla-TEM), KPN (Klebsiella pneumoniae ATCC 70063), CN (Negative Control). (B) Conventional PCR assay to confirm bla-SHV.

Bacteria carrying bla genes were isolated from all sampling sites, mainly from the Municipal Hospital and the WWTP site. The information on the relationship between the bla gene variants detected in the isolated bacteria and their potential sources of pollution is presented in Table 2.

Table 2
Distribution of bla gene variants by sampling site and isolated bacterial colonies along the Tipitapa River.

Sampling site	Bacteria isolated	bla variant detected	Economic activity	Potential source of pollution
Mouth of Lake	Aeromonas veronii	bla-SHV-24	Informal trade	Urban anthropogenic activity
			Pig farms
			Fishing
Xolotlán	Aeromonas veronii	bla-SHV-24,bla-TEM-1		Lake Xolotlán, residual water
Municipal Hospital	E. coli	bla-SHV-24	Tourism, Informal trade	Urban anthropogenic activity and recreational site.
	Aeromonas hydrophila	bla-SHV-24
				Hospital
	K. pneumoniae	bla-SHV-24
	Enterobacter cloacae	bla-SHV-24, bla-TEM-1
	Enterobacter cloacae	bla-SHV-24
WWTP	Acinetobacter soli	bla-TEM-1	Effluents of wastwater	Anthropogenic activity (urban)
	K. pneumoniae	bla-SHV-24
	Acinetobacter baumanii	bla-SHV-24, bla-TEM 1		Livestock farms
	Enterobacter bugandensis	bla-TEM-1, bla-SHV-24	Livestock
	Acinetobacter soli	bla-TEM-1
	Acinetobacter baumannii	ND
Farming area	Aeromonas veronii	bla-SHV-24	Agriculture	Urban anthropogenic activity
	K. pneumoniae	bla-SHV-13	Livestock
	Aeromonas veronni	bla-TEM-1
	Providencia rettgeri	bla-TEM-116		Livestock farms
Tisma Lagoon	Vibrio chlolerae	bla-TEM-1	Tourism	Urban anthropogenic activity and recreational site.
	Enterobacter cloacae	bla-SHV-24
	Aeromonas schubertii	bla-SHV-24
				Intensive livestock farms
				Wildlife
Human settlements	E. coli	bla-SHV-24, bla-TEM-1	Agriculture Informal trade	Urban anthropogenic activity and recreational site.
	Aeromona veronii	ND
				Fishing
Drainage basin	Klebsiella variicola	bla-SHV-13	Agriculture	Urban anthropogenic activity and recreational site.
			Livestock
			Tourism
	Aeromonas hydrophila	ND
				Livestock farms
	Aeromonas veronii	ND
				Agriculture

• ND = Not detected.

An identity of 90-100% was considered for the identification of each gene variant. Sequencing analysis showed that 21 (84%) of the isolates had four different bla gene variants distributed along the river. The most prevalent variant was bla-SHV-24, present in 10 isolates (47.62%). bla-TEM-1 was identified in 3 isolates (14.28%) along the river. Additionally, 5 isolates (23.81%) harbored both bla-SHV-24 and bla-TEM-1 genes. In a smaller proportion, bla-SHV-13 was identified in 2 isolates (9.52%). Only one isolate carried bla-TEM-116 (4.76%).

4. Discussion

Aquatic environments have been recognized as major drivers and vectors for ARG transfer in microbial communities (Hooban et al., 2020). Despite this recognition, these ecosystems in the developing world remain largely understudied due to limited research resources and effective surveillance systems. In Latin America, Brazil has been the primary contributor to the study of aquatic environment AMR (Freitas et al., 2019), followed by Chile, Mexico, and Argentina (Moreno-Switt et al., 2020).

In this context, our study provides the first report of bla gene variants in the Tipitapa River, a crucial water body used for economic activities and wastewater discharge. Additionally, it serves as a connection between the country's two major lakes, Xolotlán and Cocibolca Lake, and houses the RAMSAR site Tisma, which is essential for the conservation of flora and fauna, including migratory birds.

The Tipitapa River area features a distinctive ecosystem shaped by wind and water erosion. High-intensity agricultural practices in the surrounding region contribute to erosion, introducing mineral and nutrient loads into the river, contributing to eutrophication. The nearby Tisma lagoon serves as a reservoir for xenobiotics before the river's waters proceed to its mouth. Agriculture and livestock, including rice, bean, and wheat cultivation, and pasture irrigation, are prominent activities in this area, supporting an estimated population of around 40,204.

This region serves various purposes, including supplying water for cattle grazing, rice farming, and pasture irrigation, aiding groundwater recharge, flood control, and supporting diverse migratory bird species. While it offers vital resources like meat and fish and materials for crafts, it's crucial to acknowledge that rice farming, with its effects on water levels and agrochemical use, directly influences this site.

Our study detected four different bla gene variants in Gram-negative bacteria: bla-SHV-24, bla-SHV-13, bla-TEM-1, and bla-TEM-116 (Table 2). The presence of these variants, particularly in bacteria of global concern such as E. coli, K. pneumoniae, and A. baumanii, poses an epidemiological risk to humans and animals using the river for various purposes. As the Tipitapa River flows into Lake Cocibolca, the country's main freshwater reservoir, there is a potential risk of ARG dissemination to other ecosystems and regions of Nicaragua. Furthermore, bacteria carrying ARG can exert selective pressure on naturally occurring bacteria, exacerbating the problem.

Of particular significance is the finding that the municipal hospital and the WWTP sampling sites harbored more resistant bacteria. This can be attributed to the discharge of wastewater into the river. Previous investigations in Nicaraguan aquatic environmental sources also reported the presence of bla-TEM and bla-SHV in E. coli isolates from hospital wastewater samples in the city of León. However, in contrast to our main findings, that study reported a high prevalence of bla-CTX-M in well water and hospital wastewater samples, with bla-OXA only found in well water samples (Amaya et al., 2012). The comparison of both studies highlights the diversity of bla genes co-occurring in aquatic ecosystems.

Bacteria carrying bla-TEM-1 and bla-TEM-116 were primarily found in sampling sites close to agriculture, livestock, and urban activities, consistent with previous literature documenting the incidence of these variants in isolates from anthropogenically impacted aquatic environments (Chang et al., 2015). A review of ESBL-producing Enterobacteriaceae in freshwater environments reported that bla-SHV is commonly found in aquatic environments associated with untreated wastewater and WWTP effluents (Cho et al., 2023).

Our study is the first to report the presence of bla-SHV-13 and bla-SHV-24 variants in Gram-negative isolates from aquatic ecosystems. Notably, bla-SHV-24 was prevalent in almost all sites, including the RAMSAR sampling site Tisma lagoon, which houses wild birds, livestock farms, and various urban and recreational activities. Originally identified in clinical isolates of E. coli in Japan (Kurokawa et al., 2000), bla-SHV-24 differs by only one amino acid from bla-SHV-1, conferring high-level resistance to ceftazidime but not cefotaxime and cefazolin. Additionally, we reported the presence of bla-SHV-13 in isolates of K. pneumoniae and K. variicolla, a variant considered exclusive to clinical K. pneumoniae. bla-SHV-13 hydrolyzes cefotaxime more rapidly than ceftazidime or aztreonam. Most of the bacteria we detected are involved in human infections and are opportunistic organisms. Infections with resistant bacteria result in longer hospitalization, higher drug costs, additional diagnostic tests, and increased mortality. Antibiotic resistance genes can disseminate in the Tipitapa River/Tisma Lagoon area from different sources, including people, communities, and businesses, and through various routes like the sewage system and rivers. This can lead to the formation of antibiotic-resistant bacterial reservoirs (Figure 4, based on Wellington et al., 2013).

Figure 4
Routes of Antibiotic Resistance Gene Dispersal in the Tipitapa River/Tisma Lagoon Environment. Antibiotic-resistant bacteria with various gene variants were found near agriculture, livestock, and urban areas. Notably, new variants, such as bla-SHV-13 and bla-SHV-24, were discovered in aquatic ecosystems, with bla-SHV-24 widespread, including in the Tisma lagoon, which hosts wild birds, livestock farms, urban areas, and recreational activities. These bacteria are often associated with human infections and lead to longer hospital stays, higher treatment costs, more tests, and increased mortality. The pathways for dispersal include antibiotic release into the environment through sewage systems, with potential transportation through sewage sludge, entry into rivers, and subsequent movement through agricultural soil, surface water, and groundwater, contributing to the formation of antibiotic-resistant bacterial reservoirs.

As anticipated, the Tipitapa River exhibits high mineralization conditions that exceed previous reports (Lacayo Morales and Picado Pavón, 2022). The sampling sites at the mouth of Lake Xolotlán and the municipal hospital reported the highest EC values (Table 1). Low DO values were observed at the municipal hospital and the WWTP sampling sites, indicating environmental stressors and potential impacts on river biota. High eutrophication conditions were generally observed, indicative of elevated organic matter values. Previous studies have reported the presence of heavy metals and pesticides in Tipitapa River water (Lacayo Morales, 2020). Consequently, systematic studies are essential to assess water quality and establish policies to protect this valuable aquatic ecosystem. In light of our findings, it is advisable to establish a systematic surveillance program for antibiotic resistance gene (ARG) variants in aquatic environments, such as the Tipitapa River. This would entail regular sampling at affected sites, utilizing microbiological, biochemical, multiplex PCR, and sequencing methods for bacterial characterization. Collaboration with local healthcare facilities and wastewater treatment plants is essential to monitor the release of resistant bacteria. Additionally, it is noteworthy that the International Health Regulations (IHR) play a pivotal role in preventing the global spread of infectious diseases and in strengthening national disease prevention and surveillance systems. The proposed surveillance program would contribute to IHR implementation activities as defined by the World Health Organization (WHO, 2022).

Should additional financial resources become available, there are several ways to enhance the surveillance program. This includes expanding the program by increasing sampling frequency and extending coverage along the river. Furthermore, it would involve the implementation of advanced technologies such as metagenomic sequencing to conduct a comprehensive analysis of the river's resistome. It's essential to assemble a multidisciplinary team of experts to assess the environmental and health impacts comprehensively. Lastly, there should be a focus on developing educational programs targeting communities, farmers, and healthcare providers to reduce antibiotic misuse and enhance awareness about water quality.

Timely response requires close collaboration between scientific institutions, government agencies, and international organizations. The collected data should be shared with local health authorities and environmental agencies responsible for water quality regulation. Additionally, findings should be communicated to the World Health Organization (WHO) and relevant regional health organizations, given the potential public health implications of ARG dissemination. International scientific networks specializing in antibiotic resistance, like the Global Antibiotic Research and Development Partnership (GARDP), can play a crucial role in disseminating findings and facilitating collaborative research efforts. Public dissemination through reports, conferences, and educational campaigns is equally important to engage the local population in efforts to mitigate antibiotic resistance in aquatic ecosystems.

5. Conclusions

Urgent action is required to address the projected 10 million annual human deaths from AMR by 2050 (Murray et al., 2022). This includes studying vulnerable ecosystems, protecting aquatic environments, and raising awareness about excessive antibiotic use. Our study introduces a robust methodology with the potential for swift replication across the Central American region, thereby bolstering bacterial surveillance initiatives and enabling the geographic profiling of bla gene variants. By employing fundamental molecular techniques, we have successfully identified and characterized specific variants within the Tipitapa River. This invaluable knowledge enriches our understanding of the biochemical patterns and transmission dynamics associated with these gene variants. Further research is crucial for understanding the distribution, risk, and control of ESBL genes in aquatic environments, and we hope this research will inspire further investigations into antibiotic resistance and strategies to combat the looming AMR crisis.

Acknowledgements

The authors are grateful to Mary Rodriguez, Felipe Rodríguez, Richard J. Roberts, and New England Biolabs for reagent donations. We also appreciate the valuable input from the research team at the Molecular Biology Center CBM-UCA, including Lucía Páiz-Medina, Kenia García, Dania Gutiérrez, Fania Pérez-Mendoza, Suyen Espinoza-Miranda, and Alejandra C. Huete. Institutional support and facilities were provided by the University of Central America, UCA, Nicaragua. Dr. Huete-Pérez's work is supported by the IIE-Scholar Rescue Fund, the Marie and Felipe Educational Fund, and Georgetown University School of Foreign Service.

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Publication Dates

• Publication in this collection
  31 Jan 2025
• Date of issue
  2024

History

• Received
  19 May 2024
• Accepted
  04 Nov 2024