Aeromonas, an opportunistic pathogen present in wastewater in Tulancingo, Mexico Open Access

Wastewater is rich in nutrients such as nitrogen, phosphorus, and potassium, which can be used as a fertilizer for plants (Berbel et al. 2023); however, it can also become a serious environmental problem when released into the environment without proper treatment, as it is a major source of contamination and contains human, animal, and industrial wastes (Garcha et al. 2016). It was estimated that the volume of treated wastewater discharged into the environment exceeds 100 m³ per inhabitant/year (Sunyer-Caldú et al. 2023), with 80% of wastewater being discharged without treatment and 200 million farmers using it for irrigation (Berbel et al. 2023), meaning that flora and fauna are affected via contamination. In Mexico, there is a shortage of water, so farmers use wastewater for crop production. The main activities in Tulancingo, Hidalgo, are irrigating agriculture and intensive livestock raising, with cheese production being the most important. Ninety-seven percent of the land is used for crops and is supplied by a network of canals from the Tulancingo River (Torres et al. 2020). In a study conducted in 2014 in the same study area, they found that the maximum permissible limits for fecal coliforms were exceeded, which led to gastrointestinal infection in children from families exposed to these wastewaters (Cifuentes et al. 1993). Other authors identified Escherichia coli and Salmonella as the causative agents (López et al. 2009). Wastewater in Tulancingo is used in the cultivation of coriander (Coriandrum sativum L.), green tomato (Physalis ixocarpa Brot.), zucchini (Cucurbita pepo L.), and lettuce (Lactuca sativa L.) (Hernández-Acosta et al. 2014) and is therefore considered a potential health risk for all those who have direct contact (producers or consumers) with these products. The genus Aeromonas is mainly associated with aquatic environments, has the ability to survive chlorinated, nutrient-poor waters, harvesting systems (Gray et al. 2023), surface water, groundwater, drinking water, bottled seawater, irrigation water, and sewage water (Fernández-Bravo & Figueras 2020). In addition, Aeromonas is mentioned in the list of candidate water contaminants from the US Environmental Protection Agency as a potential pathogen that can affect human health, causing outbreaks, especially in countries where basic sanitation is poor (Carusi et al. 2024). This is also considered an opportunistic pathogen that can cause serious infections in immunocompetent and immunocompromised patients (Zepeda-Velázquez et al. 2023), as the ingestion of water and food contaminated with as little as 3 log CFU (colony forming units) g⁻¹ of Aeromonas spp. can cause local or systemic infections, intestinal and extra-intestinal diseases and/or syndromes ranging from mild conditions such as acute gastroenteritis, skin infections, and soft tissue infections to life-threatening conditions such as severe gastroenteritis, septicemia, necrotizing fasciitis, myonecrosis, and bacteremia (Janda & Abbott 2010; Carusi et al. 2024). Infections can also be observed in the skin, soft tissue, intra-abdominal regions, respiratory tract, urogenital tract, and eyes (Fernández-Bravo & Figueras 2020). Aeromonas spp. are also responsible for infections in fish, vertebrates, and invertebrates (Janda & Abbott 2010), with this genus being one of the most representative genera in wastewater (Fernández-Bravo & Figueras 2020; Aoki et al. 2023). Therefore, the objective of this study was to identify the presence of the genus Aeromonas in wastewater from one of the drainage networks belonging to the Rio Grande de Tulancingo de Bravo in the State of Hidalgo, Mexico, because wastewater becomes a vehicle for transferring pathogens to different sources of application, either directly on leaves or fruit or indirectly through splashing, which can become a major public health problem.

Twenty-five samples were taken in accordance with the Mexican Official Norm NOM-001-SEMARNAT-2022 (SEMARNAT 2022), which establishes the permissible limits of pollutants in wastewater discharges into receiving bodies owned by the nation. A sample was taken in the morning to obtain samples that represented actual conditions; if taken at another time of the day, the water flow was affected by various human activities in the area. Sampling was performed weekly for 6 months (June–November) at 9:00 am. The following steps were carried out: when sampling began, the sample container was rinsed repeatedly before being introduced into the water discharge. The sample was transferred from the sampling container to the container for the sample. The sample was taken where there was the highest water flow the walls or the bottom were not touched, and samples were not taken if there was rain due to the dilution, as this could influence the results; the volume of each sample was 500 mL, even though we worked with smaller volumes in the laboratory to provide representative data.

Isolates were seeded on trypticasein soy agar (TSA) and incubated at 30 °C for 24 h; subsequently, a colony was suspended in 200 μL of sterile distilled water and incubated at 100 °C for 10 min before being used directly in PCR reactions (Reyes-Rodríguez et al. 2015).

The suspected isolates of Aeromonas spp. were confirmed using the glycerophospholipid-cholesterol acetyltransferase (gcat) gene PCR because this is a gene that is capable of identifying genus (Chacón et al. 2002; 2003) (Table 1). The PCR reaction included 5 μL 10× PCR regulator (50 mM KCl, 75 mM Tris-HCl (pH 9.0)), 1.5 mM MgCl₂, 1 μL 10 mM mixture of dNTPs, 0.5 μL Taq DNA polymerase (5 U/μL) (GoTaq^® Flexi DNA Polymerase, Promega), 1 μL 10 mM of each primer, and 5 μL of DNA, adjusted to a final volume of 50 μL. Amplification of the rpoD gene in isolates that were positive for Aeromonas was performed using the primers described by Yamamoto & Harayama (1998) (Table 1) because this gene is able to identify species of Aeromonas (Chacón et al. 2002). The isolates obtained with similar morphology to Aeromonas in ADA medium were not analyzed with the gcat gene; they were analyzed with the rpoD gene for molecular identification. Touchdown polymerase chain reaction (PCR) is a method to decrease off-target priming and hence to increase the specificity of PCRs (Soler et al. 2004). The PCR reaction included 5 μL 10× PCR regulator (50 mM KCl, 75 mM Tris-HCl (pH 9.0)), 2.0 mM MgCl₂, 1 μL 10 mM mixture of deoxynucleotide triphosphates (dNTPs), 0.5 μL Taq DNA polymerase (5 U/μL) (GoTaq^® Flexi DNA Polymerase, Promega), 1 μL 10 mM of each primer and 5 μL of DNA, adjusted to a final volume of 50 μL. The presence of amplicons in 1.5% agarose gels was verified using Tris-borate-EDTA (TBE) buffer and staining with ethidium bromide. A DNA ladder ranging from 100 bp to 1,500 bp (Promega) was used to identify the band size. Purification of the amplified product was performed using the Wizard^® SV Gel and PCR Clean-Up System kit (Promega) and sequenced using the Sanger method. The obtained sequences were assembled with the computer program DNAstar SeqMan (Lasergene). Nucleotide sequences were aligned using the Clustal W program, including the published sequences of all the reference strains of the currently identified Aeromonas species (Reyes-Rodríguez et al. 2019).

According to the WHO (2006) guidelines for the safe use of wastewater, excreta, and graywater, wastewater is increasingly used for agriculture, and population growth influences more wastewater discharge, so urban agriculture plays a very important role in food supply, and municipal wastewater could become the only source of water for many farmers due to its scarcity, population growth, urbanization, and climate change. The Food and Agriculture Organization of the United Nations mentions that treated wastewater with low microbial and chemical contamination can be used directly or indirectly for aquaculture, agricultural, industrial, and domestic purposes (FAO 2013); however, these wastewaters and wastewater treatment plants have favorable conditions such as aeration and optimum temperature and nutrient contents for bacterial proliferation (Majeed et al. 2023). In communities with high levels of poverty that depend on aquatic environments to meet their water needs, the aquatic environments are critical points for the spread of bacteria due to pollution caused primarily by anthropogenic activities (Govender et al. 2021). In this study, the point where the sample was taken was selected because it carries the residual waste from animal production units, the textile industry, hospitals, and residential areas. All of this occurs within a range of 5 km because it is a relatively small area compared to the Tulancingo Valley, which comprises 10 municipalities (1,054 km² and partially includes the municipalities of Tulancingo de Bravo, Metepec, Acatlán, Cuautepec de Hinojosa, Santiago Tulantepec de Lugo, Singuilucan, Huasca de Ocampo and small portions of the municipalities of Acaxochitlán, Tenango de Doria, and Agua Blanca de Iturbide). In Tulancingo there is a disorderly hydraulic infrastructure, which has resulted in a significant accumulation of waste in the hydraulic network. It is estimated that 280 L/s of wastewater were generated and that it was discharged into the rivers in the area. It also joins with rainwater collection because it does not have an independent drainage system, so there is a significant concentration of water in the areas where the sampling was performed, causing a severe environmental impact. Because of the concentration of water in Tulancingo, eight water treatment plants were installed at one time, but only one is in operation. In the municipality, based on regulatory policies, the regulation of land use for residential areas has begun to be implemented, due to the proliferation in high-risk areas. A clear example is the sampling area where to the north (2 km) is the nearest hospital, to the south and east (1 km or less) are residential areas and livestock production units, and to the west (1 km or less) is an industrial area, several production units, and residential areas. All this information is of utmost importance because Tulancingo has suffered several floods over the years. According to Herrera et al. (2018) it is due to meteorological phenomena or extreme rainfall, in addition to the fact that there is the interaction of waves from the east with flows from the west with tropical cyclones in conjunction with the fact that Tulancingo receives runoff from the highlands. An example is what happened in the year 2024, where 350 m from the sampling area is located the nearest residential area, where the wastewater came out of the water registers and entered the houses that were close to the water channel, reaching between 5 and 50 cm in height. In addition, these waters reach agricultural cultivation areas. From the perspective of one health, direct contact with this wastewater from livestock production units, factories, hospitals, and residential areas can generate major environmental problems in addition to becoming a critical reservoir for the genus Aeromonas, putting at risk the health of the inhabitants exposed. From the approach of one health, the ecosystem, people, and animals are closely related so that the presence of these opportunistic pathogens puts at risk the appearance of diseases.

In Tualancingo, Hidalgo, Mexico, Aeromonas is considered an opportunistic pathogen, with 36 species having been reported; however, 19 of these are considered pathogens causing disease in humans and animals (Carusi et al. 2024) and have the ability to grow in seawater, brackish water, freshwater lakes, ponds, rivers, municipal drinking water, bottled mineral water, wastewater effluent, and hospital wastewater effluents (Majeed et al. 2023).

It was mentioned that the infectious dose from ingesting contaminated food and water is probably as low as 10³ CFU of Aeromonas spp. and can cause local and systemic infections (Carusi et al. 2024); their abundance is often correlated with fecal indicators, particularly in environments impacted by sewage; however, Aeromonas is not classified as being of fecal origin (Milligan et al. 2023). It is also considered in the connections between sanitary waste systems, aquaculture production, and natural aquatic environments (Jones et al. 2023).

In this study, the identity of the species with the rpoD gene was calculated using the type of strain, where Aeromonas caviae was identified in 43.2%, Aeromonas media in 29.5%, Aeromonas hydrophila in 11.3%, Aeromonas salmonicida in 9.1%, and finally Aeromonas allosaccharophila, Aeromonas lusitana, and Aeromonas veronii in 2.3% each (Figure 3, Table 2).

In this study, A. caviae and A. hydrophila were identified; Nowrotek et al. (2021) consider them to be the most pathogenic species, and it is estimated that 85% of the infections were caused by A. caviae, A. hydrophilia, and A. veronii, which generated infections in the skin, soft tissues, and gastrointestinal tract. A. caviae has been isolated from municipal wastewater from Warmia and Mazury, Poland (Hubeny et al. 2019). Govender et al. (2021) identified A. hydrophila, A. sobria, A. caviae, A. veronii (10%), and other Aeromonas spp. (8%) from wastewater in Durban, South Africa.

In Tokyo, they identified A. hydrophila and Aeromonas dhakensis from hospital wastewater (Ota et al. 2023); in wastewater from Kokšov-Bakša, Slovakia, they identified 228 isolates of Aeromonas spp. (Kisková et al. 2023). In Japan, in sewer lines combined with domestic and industrial wastewater and rainwater runoff, they found A. caviae in 33.3% (Tanabe et al. 2023). In municipal wastewater from the metropolitan city of Tshwane, South Africa, they found 2.8% Aeromonas species, being the most prevalent enteric pathogen, with a relative abundance ranging from 0.7 to 5.3% (Poopedi et al. 2023).

Aeromonas are natural inhabitants of aquatic environments and have been detected in samples obtained from different sources (Pessoa et al. 2022); these microorganisms can be used as ecological indicators of water pollution (Grilo et al. 2021). In a study conducted in the Erie Canal, they identified A. allosaccharophila, A. bestiarum, A. hydrophila, A. jandaei, A. sobria, and A. veronii and stated that the contamination was due to tributaries from sanitary sewer overflows, faulty septic systems in the lot, municipal sewer conveyance systems, and stormwater discharges that reach Lake Erie and can serve as a source of bacteria of human origin (Skwor et al. 2014).

Twenty species of Aeromonas have been identified in the Atlantic region; however, A. veronii, A. hydrophila, and A. jandaei predominated in freshwater rivers, tidal brackish rivers, wastewater effluents, and production unit ponds, accounting for ∼63% of the isolates (Martone-Rocha et al. 2010). It was reported that microbiologically contaminated irrigation water is the cause of food and waterborne outbreaks (Solaiman & Micallef 2021), whereby the persistence of pathogenic bacteria and other organisms in wastewater can spread to humans through the food chain (Martone-Rocha et al. 2010).

In a study conducted in Australia, Aeromonas was identified in patients with deep and superficial infections in 63% (201/317) and 80% (375/468), respectively (Campbell et al. 2024).

In this study, we were also able to identify one isolate of Pseudomonas sp. Irchel, Acinetobacter towneri, Providencia huaxiensis, and Enterobacter hormaechei subsp. Hoffmannii (Table 3). Pseudomonas sp. Irchel causes an infection called chlorinquia or green nails and is found in damp places (Baran & Richert 2024). These bacteria are used in groundwater bioremediation because they are capable of simultaneous nitrification and denitrification under aerobic conditions (Liu et al. 2024).

Acinetobacter towneri was identified in this study; however, this genus is ubiquitous in the natural environment and has become an important opportunistic pathogen. Acinetobacter towneri has been identified in swine (Li et al. 2022), in a municipal landfill leachate treatment bioreactor (Szilveszter et al. 2023) and in hospital wastewater (Jiang et al. 2019); it was involved in nosocomial infections (Li et al. 2022) and has been identified in patients with recurrent urinary tract infections and chronic appendicitis (Zou et al. 2017).

Providencia huaxiensis has been identified in marine fish sediment (Selepe & Maliehe 2024) and in human rectal swabs (Hu et al. 2019); this is also used in the bioremediation of wastewater (Selepe & Maliehe 2024). Finally, Enterobacter hormaechei subsp. hoffmannii has been identified in patients with urinary tract infection, in the sputum and blood cultures of a patient with pneumonia, from the drainage effluent of a patient with post-surgical infection (Umeda et al. 2024) and in artisanal goat cheeses (Nelli et al. 2023).

Crop contamination can occur at any stage of the food production chain, including upstream and downstream processing stages; for example, pre-harvest contamination can occur through contaminated irrigation water, workers, inadequate implementation of sanitation, or fecal contamination (Zarkani & Schikora 2021).

The use of reclaimed water derived from treated wastewater for agricultural irrigation is gaining attention due to rapid urbanization, especially in developing countries. With increasing global water scarcity, it was estimated that 40.7 × 10⁹ m³ year⁻¹ of treated wastewater is intentionally reused for human purposes; however, special attention must be paid to the inadequate removal of pathogenic microorganisms (Aoki et al. 2023).

The discharge of pollution and wastewater due to human activities directly or indirectly into receiving water resources, especially rivers, is one of the most dangerous threats to water resources (Horn et al. 2022). The direct and indirect reuse of partially treated or untreated urban wastewater in irrigation-dependent urban and peri-urban food production systems is now common practice in many countries because of the access to irrigation water, but the urban contamination of river ecosystems jeopardizes these advantages. The discharge of untreated wastewater and the consumption of food sources contaminated by polluted river water due to open defecation have led to instances of Aeromonas-related gastroenteritis occurring worldwide, with cases being most common in developing countries; waterborne diseases are expected to increase with climate change and the growing world population (Poopedi et al. 2023).

The microbial quality of wastewater in Tulancingo, Hidalgo, was affected by agricultural and industrial waste, waste from wildlife and farms, and discharge from municipal sewer lines. The World Health Organization (2022) indicates that the role of contaminated water used in the production of vegetable crops as a vector of transmission of these pathogens to humans is unclear. Suslow et al. (2003) indicate that water contaminated with human or animal waste can introduce pathogens to plants during pre-harvest and post-harvest activities.

Pachepsky et al. (2011) stated that it was difficult to prove that irrigation water causes foodborne illness because it requires the same pathogenic strain to be isolated from the patient, the product and the irrigation sources. Therefore, it can only be inferred based on circumstantial or subjective evidence.

Foodborne disease outbreaks in fruit and vegetable crops are frequently associated with irrigation water; in a study conducted on romaine lettuce leaves, there was a certainty that Aeromonas can be transferred from irrigation water to fresh vegetables and can act as a vehicle for the transmission of infections. Furthermore, it was known that hydroponically grown produce internalize bacteria through its roots (Dorick et al. 2023), which gives us some idea of the potential for persistence in food processing environments and the possible risk of the contamination of fresh crops, indicating a possible risk to food safety (Solaiman & Micallef 2021).

Rivers, ponds, and reclaimed water sources are the main reservoirs of Aeromonas species known to cause disease, raising the possibility of these species entering the food production chain and persisting for prolonged periods. Wu et al. (2023) mention that wastewater from production units, agricultural lands, housing environments, pharmaceutical factories, and hospitals normally accumulates in lakes, rivers, oceans, and soils, so the bacteria present can continuously contaminate the environment; from this perspective of One Health, water in the natural environment is considered a reservoir of Aeromonas, which is a bacterium that can cause disease.

In Mexico, digestive diseases are among the main ones in the population, with between 5 and 6 million new cases reported each year (Olaiz-Fernández et al. 2020); however, the Mexican Ministry of Health indicates that 94% of diarrheal infections do not have a determined etiological agent and states that demographic, economic, social, and educational conditions can influence the presentation of diseases (Secretaria de Salud 2021). Menchaca-Armenta & Gutiérrez-Jaimes (2022) reported Aeromonas to be among the main pathogens associated with infectious diarrhea in Mexico, with an estimated prevalence of diarrheal diseases in children under 5 years of age of 11.8% nationally and 13.7% in Hidalgo.

Eluma et al. (2023) mention that Aeromonas was related to outbreaks of gastroenteritis in Nigeria due to vegetable consumption. These crops were contaminated by direct contact with irrigation water or by pesticide or fertilizer diluents, finding a prevalence of 25.25%.

The contamination of vegetables can occur at various stages; however, irrigation with inadequately treated wastewater is one of the main causes of contamination. In China, Aeromonas was identified in lettuce, parsley, and watercress, with a level of 19.1%, and it was stated that worldwide morbidity due to the consumption of fruits and vegetables has caused enteric bacterial infections, especially in poor environmental conditions (Elsafi et al. 2024).

In a retrospective study in India, in 1595 suspected cholera patients, 3.1% (50/1595) were found to be due to Aeromonas, causing severe dehydration, typical cholera stools, or bloody stools. The authors indicate that this may have been due to contact with food contaminated with irrigation water (Mohan et al. 2017).

In a study conducted in China, it was reported that, out of 5,069 samples of patients with diarrhea and water samples, 4.5% (193/5,069) were due to the presence of this etiological agent; however, they indicate that the main sources of contamination in infections caused by Aeromonas were aquatic environments (Li et al. 2015). It is said that soil physical qualities, fertility, soil structure, and organic matter content can be improved by irrigation with wastewater that has been properly treated; however, there are reports that Aeromonas is present even after wastewater treatment (Jones et al. 2023). Skwor et al. (2014) mention that Aeromonas species are found in chlorinated wastewater effluents due to chlorine resilience (1.2 mg/L), so they suggest implementing other treatment methods such as ultraviolet radiation, ionization or ultrasound. Therefore, the isolation of this genus in wastewater suggests that these serve as reservoirs because they are not adequately treated, so this water could be considered a reservoir for this pathogen and a potential route for its transmission to humans and animals, making it an important health risk. Thus, in this study, we can assume that the presence of Aeromonas in wastewater is a risk factor because it is used in the irrigation of various plant products. Having direct contact, together with inadequate handling, could be the cause of the presentation of digestive disease; however, there is no surveillance of this pathogen in Mexico, so in cases of diarrhea where the etiological agent is not identified, Aeromonas is probably the cause.

Aeromonas has been identified in diarrheal disease in Africa, Asia, Europe, Oceania, and the Americas, especially in Brazil, Cuba, Peru, and the United States, with very few outbreaks described (Figueras & Ashbolt 2019). It was reported that there has been non-compliance with Koch's postulates (Teunis & Figueras 2016), so it is difficult to determine the association with disease presentation. However, in a study carried out in Spain, Aeromonas was epidemiologically linked in water and vegetable samples (lettuce, tomato, and parsley) (Latif-Eugenín et al. 2017). This indicates that irrigation water contaminates fruits and vegetables and is therefore a vehicle of transmission. In addition, it has been possible to identify the same genotype in patients with diarrhea due to water and food transmission (Fernández-Bravo & Figueras 2020). Latif-Eugenín et al. (2017) mention that Aeromonas has the ability to regrow. A study by Jjemba et al. (2010) mentions that Aeromonas and Pseudomonas were effectively eliminated from wastewater by different disinfection mechanisms such as chlorine, ozone, and ultraviolet radiation; however, they regrew at a higher concentration in water distribution systems. This is because the effluent from properly treated wastewater passes into the distribution systems, and when the water is rechecked, Aeromonas is again identified because these systems are contaminated. Which puts their presence at risk, either in wastewater, because nutrients and temperatures make a favorable environment for the survival of these opportunistic pathogens.

In Mexico, Aeromonas is not considered a notifiable microorganism but is listed as a pathogen that causes secondary contamination in the production chain. It can cause disease in moderate to low doses (10³–10¹⁰) (Figueras & Ashbolt 2019). Therefore, Aeromonas should be considered a cause of gastroenteric outbreaks. Therefore, it is necessary to integrate all the fields, taking into consideration the social, cultural, economic, and environmental areas of the study area through the use of a comprehensive approach to risk assessment and management, ranging from the generation, use, or contact of wastewater to the consumption of the product.

Aeromonas spp. are widely distributed in the environment; however, aquatic environments are the main sources. These species are capable of causing infections in humans due to the ingestion of food or water contaminated with these bacteria or by direct contact with water. In this study, the presence of Aeromonas was identified, a genus that is known to cause intestinal and extra-intestinal diseases; it is known that these species can enter the food production chain, coupled with the fact that these bacteria can resist treatment processes, which represents a potential contamination route. Therefore, hospital, industrial, animal production, and domestic wastewaters are major spreaders in aquatic environments and are capable of colonizing sewage treatment plants and, consequently, contaminating agricultural products intended for human consumption. However, the presence of Aeromonas in wastewater cannot be underestimated, and it is already considered an opportunistic pathogen; therefore, its presence should be considered a threat to health, and it is necessary to develop strategies to monitor its presence in aquatic environments, more specifically through epidemiological studies whose objective is to evaluate the health risks associated with the use or contact by comparing the level of disease with the exposed population.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.