Effect of Two Types of Wastewater Treatment Plants on Antibiotic Resistance of Fecal Coliform

Abstract

The existence of fecal coliform microorganisms (FCs) resistant to antibiotics in the domestic wastewater of an urban and semi-urban locality was determined, along with the effect of two types of treatment plants for wastewater on the resistance of coliform, an aerated lagoon (AL) and a stabilization lagoon (SL). Samples were taken from the affluent and effluent of each treatment plant. FC content, pH, electrical conductivity, dissolved oxygen, total solids, total volatile solids, and several types of ions were determined. Resistant FCs were quantified by plate count in bright green bile agar with ampicillin, amoxicillin, sulfamethoxazole-trimethoprim, amikacin, gentamicin, cefixime and their mixtures. The isolated strains were evaluated against other antibiotics using antibiograms. The relationship between the variables was validated with an analysis of variance factorial design, and Fisher’s means test (α = 0.05) and Pearson’s correlation were used to establish it. The community that presented more resistant FCs was the urban one, but when the wastewater passed through the systems of AL and SL, this fact changed. The resistance of the FCs to ampicillin, amoxicillin, trimethoprim, sulfamethoxazole and cefixime was higher in the SL, with values of 67, 48, 2 and 25.8%, while those for the AL were 20, 13, 22 and 5.3%, respectively.

1. Introduction

Antibiotics are probably the most successful family of drugs used in improving human health, and are even used to prevent disease in agriculture and livestock [1]. However, the intensive use of antibiotics in human health, veterinary medicine and agriculture results in the continuous release of these substances into the environment, raising concerns about the development of both antibiotic resistance genes and resistant bacteria, and reducing the therapeutic potential of these drugs [1,2]. The emergence of antibiotic-resistant bacteria is a growing global public health problem [3]. The World Health Organization has identified antimicrobial resistance as a major threat to human health [4].

Large amounts of antibiotics are released into municipal wastewater through incomplete metabolism or the disposal of unused antibiotics, exposing bacteria to them and promoting resistance [4,5].

Domestic wastewater treatment has been shown to reduce the levels of both antibiotic-resistant and antibiotic-naive bacteria, and to alter the proportion of resistant bacteria by increasing their numbers in treated water [5]. This is because the aquatic environment is a reservoir for both resistance genes and resistant strains [6], so domestic wastewater treatment plants (WTPs) are considered a reservoir for the development and spread of antibiotic resistance [7]. This may be due to three reasons: (1) antibiotic residues and substances with the potential to exert selective pressure, and carrying resistance genes, are discharged into the sewerage system; (2) the conditions provided by these systems may favor the selection or horizontal transfer of resistance genes [8]; and (3) as observed worldwide, there is a higher percentage of resistant bacteria at the end of treatment than at the beginning [2,4,7,8,9].

Another factor that affects the durability of FCs is the type of treatment plant used to carry out depuration. The most used are of the aerobic type, which can be mechanized or not, the difference being the hydraulic retention time (HRT) and the way the air enters the system. In the first case, a mechanized plant, the HRT of the plant is 3 to 5 days, and it uses pumps that aerate the lagoon where the water is stored. In non-mechanized plants, the HRT of the water can be up to 22 days, and air enters through natural diffusion effects that are influenced by the area and depth of the lagoon [9,10].

Some studies have suggested that multidrug-resistant and susceptible bacterial populations are not affected by the wastewater treatment process through which they pass [5]. However, others suggest that the different process units of the WWTP influence the resistance presented by different microorganisms [10]. This is because the type of treatment can influence the selective pressure for antibiotic-resistant microorganisms, where these genes have been found to be closely related to stress genes [3,4]. Munir found that compared to conventional WWTPs using activated sludge or aerated lagoons, WTPs with a membrane disinfection system could further reduce the concentrations of both antibiotic-resistant microorganisms and genes.

In the state of Durango, México, 80% of the WTPs are stabilization lagoons operating with non-mechanized systems, 95% of which discharge their water into the rivers of the area, and this has been found to cause undesirable changes in the corresponding microbial composition [11]. In addition, it can turn these systems into eutrophic systems. On the other hand, the water (1500 L/s) of the East WTP, the largest in the city of Durango, State of Durango, México, is discharged into agricultural lands in the peri-urban region, and although wastewater treatment is a basic public service, it is important to know the risks.

Therefore, the objective of this work was to compare the effluent quality of two types of domestic wastewater treatment plants, an aerated lagoon (AL) and a stabilization lagoon (SL), and their effect on the amount of fecal coliform microorganisms resistant to six of the most used antibiotics in the city of Durango. In addition, two types of communities were studied: one urban (Durango City) and the other semi-urban (10 km from Durango City).

2. Materials and Methods

The resistance of fecal coliform (FC) microorganisms to 6 types of antibiotics (the most common in health centers in both urban and semi-urban areas) and their mixture was studied. Also, we wanted to determine if said systems, a mechanized aerated lagoon and a non-mechanized stabilization lagoon, influenced the resistance exhibited by the FCs after treatment.

2.1. Selection of Water Treatment Plants

The wastewater treatment plants (WTPs) involved were of the mechanized (aerated lagoon) and non-mechanized (stabilization lagoon) types. In both cases, the contents of fecal coliform microorganisms in the influent and effluent were determined, as well as the resistance of the microorganisms to 6 commonly used antibiotics and their mixtures.

Physicochemical parameters were also determined, and the study areas and characteristics of the WTPs are described here. Likewise, the populations they serve, one semi-urban (stabilization lagoon) and the other urban (aerated lagoon), were compared, since there were different consumption habits and degrees of access to medicines between them.

2.2. Characteristics of the Treatment Plants

The aerated lagoon (AL) WTP serves a community of 616,068 inhabitants and is located at 24°01′32.38″ N, 104°36′19.26″ W. It treats an effluent of 1500 L/s and flows into the river “Tunal”, which uses its water for irrigation. The stabilization lagoon WTP serves a community of 2230 inhabitants located at 24°08′34.66″ N, 104°34′50.37″ W, 10 km from the city of Durango, so it is not considered completely outside the influence of the urban area and uses the treated water for the irrigation of grasslands. This can be seen in Figure 1.

2.3. Determination of the Physicochemical Parameters

Samples were taken four times in the case of the aerated lagoon and three times in the case of the stabilization lagoon, each time in duplicate (with a 15-min interval between them) at the inlet and outlet of each system.

For the determination of physicochemical variables, the samples were collected in 1 L plastic bottles. Separate samples were collected in 500 mL flasks for the determination of NH₃/NH₄+, which were immediately fixed with 1 mL sulfuric acid. The samples were then transported to the laboratory in cool boxes.

pH, electrical conductivity in µS/cm (EC), water temperature (°C) and dissolved oxygen (DO) were measured in situ at a depth of 30 cm below the water surface using a HACH HQ40 multi-parameter meter. The instrument was calibrated according to the manufacturer’s specifications.

In the laboratory the following was determined, in mg/L: total solids (TS), total volatile solids (TVS), fluoride (F⁻), chloride (Cl⁻), soluble or reactive phosphorus (PO₄³⁻), nitrate (NO₃⁻), and ammonia compounds (NH₃/NH₄⁺). We used a Metrohm™ ionic chromatograph 883 model Basic IC Plus (Metrohm, 1943, Herisau, Switzerland).

The toxicity of the ammonia compounds (NH₃/NH₄⁺) is attributed to the unionized (NH₃) chemical species present in aqueous solution [12]. Since the percentage of total ammonia present as unionized ammonia (NH₃) is so dependent on pH and temperature, in this work, it was determined using the following equations and reported asNH₃:

$$pKa=0.00901821+2729.92/T$$

(1)

where T = temperature in K; absolute zero = −273.15 °C.

$$f=1/[{10}^{\left(pKa-pH\right)}+1]$$

(2)

where f = fraction of total ammonia that is unionized; pKa = dissociation constant from Equation (1).

The sampling, analysis, transport and storage of the samples were carried out according to the criteria established in the Standard Methods for the Examination of Water and Wastewater [13]. The average values of the effluent and affluent data of the sampling were compared with the permissible limits for irrigation and aquatic life, according to the official Mexican standard [14] and ecological criteria of 1989 [15].

2.4. Determination of Antibiotic-Resistant Coliform Microorganisms

The plate count method was performed using brilliant green bile agar (BD Bioxon, Estado de México, Mexico) to determine fecal coliforms, and the same culture medium was used to test for bacteria resistant to the following six antibiotics using the poisoned agar technique: ampicillin (AMP 10 µg/mL), amoxicillin (AMX 25 µg/mL), sulfamethoxazole-trimethoprim (SXT 25 µg/mL), amikacin (AMK 30 µg/mL), gentamicin (GEN 10 µg/mL) and cefixime (CFM 5 µg/mL). We also applied their mixture in the same concentrations to look for organisms that could be multi-resistant (MULTI). It is worth mentioning that the antibiotics were provided by the health center, since they are the most widely distributed among the population.

These antibiotics were solubilized in a gastric solution, a modification of that reported by [16], which consisted of a mixture of sodium chloride (NaCl) at a concentration of 0.05 N and hydrochloric acid (HCl) at 0.07 N to achieve greater solubility. This resulted in 7 treatments and one blank without antibiotics. This procedure was performed for both the inlet and outlet water. The plates were incubated at 37 °C for 24 h. This procedure is shown in Figure 2. This was done for both inlet and outlet WTP samples.

2.5. Determination of Multi-Resistant Strains

Subsequently, fecal coliform strains were selected, isolated and identified by biochemical tests using the API20E system from each site studied and from each antibiotic treatment. Once isolated and identified, their resistance to several antibiotics was tested in Mueller–Hinton agar against ceftriaxone (CRO, 30 µg), cefuroxime (CTX, 30 µg), cefepime (FEP, 20/10 µg), ciprofloxacin (CIP, 5 µg), amoxicillin/clavulanate (AMC, 30 µg), penicillin (P, 10 µg), vancomycin (30 µg), imipenem (APM, 10 µg), linezolid (LZD, 30 µg), piperacillin/tazobactam (TZP, 100/10 µg), levofloxacin (LVX, 5 µg), and ampicillin (AM 10 µg) with a BBL Sensi-Disc™ to determine whether organisms that had already shown resistance to one antibiotic also showed resistance to other types of antibiotics.

Plates were incubated at 37 °C for 24 h. The results were read by measuring the diameters of the growth inhibition halos that appeared around the paper discs and compared with the parameters specified by the manufacturer.

2.6. Calculation of the Proportions of Antibiotic-Resistant Microorganisms

The proportion of antibiotic-resistant coliforms (% CRA) was determined using the following formula:

$$\%ARC={\displaystyle \frac{RM\times 100}{TC}}$$

(3)

where %ARC—percent of antibiotic-resistant coliforms; RM—antibiotic-resistant microorganisms count for each antibiotic/treatment; TC—total count of coliform microorganisms. This value was taken from the plates without any antibiotic added.

2.7. Information Analysis

Microsoft Excel software (Microsoft 365 version number 2407 compilation 17830.20138) was used for the organization of the information and the description of the results found. The data obtained from the samples were statistically analyzed with STATISTICA^® version 7 software. For this, analysis of variance (ANOVA) and Fisher’s tests were used to validate significant differences between the resistances to the different antibiotics applied to the FC of the two types of WTP. For the correlations between resistance and physicochemical parameters in each WTP, Pearson’s correlation was used. Everything was tested at the level of α = 0.05.

3. Results

3.1. Performance of Treatment Plants

In the water samples taken from the affluent and effluent of the two domestic wastewater treatment plants (WTP), their FC content was studied in addition to some physicochemical factors, such as pH, electrical conductivity in µS/cm (EC), and water temperature (°C), as well as dissolved oxygen (DO), total solids (TS), total volatile solids (TVS), chlorides, nitrites, nitrates, phosphates, sulfates and ammonia in mg/L. In this work, the WTPs tributaries are designated as urban and semi-urban according to their origin, and the effluents are designated as aerated lagoons and stabilization lagoons according to the type of WTP; see Supplementary Material Table S1.

The differences found were validated by means of a factorial ANOVA and a Fisher’s means test, all at a probability of α = 0.05 (see

Table 1. Average affluent and effluent with n = 8 from the WTP aerated lagoon (AL) and n = 6 from the stabilization lagoon (SL). The same letter means that there is no statistical difference between the samples. For comparison, the limits in the parameters for river and irrigation water, for which that mark of the NOM-001 and ecological criteria are also shown. If marked in bold and underline, it means they are out of limit.

Sample	pH	EC	T	log FC	DO	TS	TVS
		µS/cm	°C	UFC/mL	mg/L
URBAN AFFLUENT	7.5a	729a	25.0	5.7b	0.2a	613a	274b
AL EFFLUENT	7.3a	761a	21.4	4.2a	1.1b	519a	202a
SEMI-URBAN AFFLUENT	7.2a	1615b	21.5	5.7b	3.2c	1197b	447d
SL EFFLUENT	8.0b	1696b	18.4	5.0a	0.4a	1175b	400c
River	6.5−8 *	N.C.+ *	N.C.+ *	N.C.+ *	5 *	N.C.+ *	N.C.+ *
Irrigation	6−9+	1000+ *	N.C.+ *	2+ *	N.A.+ *	N.C.+ *	N.C.+ *
Sample	F−	Cl−	NO2−	NO3−	PO43−	SO42−	NH3
	mg/L
URBAN AFFLUENT	3.0a	35a	0.5a	0.3a	2.9a	70a	0.493a
AL EFFLUENT	3.0a	47a	0.3a	0.3a	2.9a	76a	0.256a
SEMI-URBAN AFFLUENT	6.1b	101b	0.6a	0.4a	11.6b	48a	1.865c
SL EFFLUENT	5.9b	168c	0.2a	0.4a	13.2b	82a	0.584b
River	1 *	250 *	0.3 *	90 *	0.1 *	0.005 *	0.06 *
Irrigation	1 *	147 *	N.C.+ *	N.C. *	N.C. *	130 *	N.C. *

Notes: N.C. = not contemplated within the Mexican regulations but know to alter water quality; N.A. = does not apply; + = referred to in the Mexican Official Norm NOM-001; * = referred to in the ecological criteria of water quality.

). Statistically significant differences were found between the affluent and effluent of the two systems in pH, EC, ST, STV, F⁻, Cl⁻, PO₄³⁻ and NH₃; the FC content is the most important parameter in this work. It showed significant changes in the inlets and outlets, but not in the aeration lagoon system with respect to the stabilization lagoon system, where the contents were the same. The effluents of both lagoon systems did not comply with the contents standards for fluorine, phosphates, ammonia and FC for discharge into rivers and irrigation; they do not comply with what is required for irrigation, because they exceed it.

The reviewed lagoon systems, which originate from different entities—an urban area and a semi-urban area—demonstrate substantial chemical alterations that persist despite purification efforts. These changes do not affect the effluent’s quality. The results show that the semi-urban system passes the EC and fluorine regulations by 1.5 and 6 times, respectively. In contrast, the urban system only exceeds the fluorine regulation by 3 times.

Neither of the effluents meets the limit values in nutrients such as phosphates and ammonia. While they do not cause problems in irrigation, they will undoubtedly cause changes in the physical–chemical properties of aquatic environments, which will have repercussions for the ecosystem in general. The most common of these repercussions is eutrophication, which sometimes is accompanied by excessive algae growth [17,18].

3.2. Fecal Coliform Antibiotic Resistance in the Populations

The FCs were tested for their resistance to the 6 most distributed antibiotics by the local health center and their mixture to see if there were any multi-resistant strains, and the effect was analyzed by community (at the entrance of the WTP) and by treatment system (at the exit of the WTP), in addition to applying Pearson’s correlation, to establish effective affinities between the revised parameters, all at a probability of α = 0.05 (see Figure 2 and Table S2).

There are significant differences (F = 4.72 m p = 0.03) in the survival presented on average by the urban community with respect to the semi-urban one. It is greater in the urban one; this could be due to the accessibility to antibiotics in an urban community compared to maybe a more limited supply of medicines in a semi-urban community.

The differences by antibiotic are shown in Figure 3 and Table S2, showing the statistical differences (F = 24.13, p = 0.000), where CFM and AMP were between communities.

Regarding the number of FCs, these were not significantly different between the urban and semi-urban communities, with an average of 505,625 CFU/mL (Table S2). As mentioned before, they were only differences between communities in the response to CFM and AMP. For the other treatments we did not find statistical differences. Also, we can see that the biggest resistance was towards AMP, AMX and SXT.

In general, the antibiotics with the least resistance in FC were AMK and MULTI, where survival did not reach 1%, followed by GEN, with 1.34% survival. In general, the community with more resistant FCs was the urban community. The percentages of survival observed for the antibiotics AMK and MULTI were significantly correlated with pH, µS/cm EC, % DO and temperature, and the remaining antibiotics did not show a significant correlation with the physicochemical parameters (see

Table 2. Pearson’s correlation between the physicochemical parameters and the % survival of FCs to antibiotics (N = 14; p < 0.05) for water by community. The values in bold and underlined were significant for the water by community.

Urban Community	TVS	Cl−	NO3−	SO42−	NH3
Total FC	−0.74	0.14	−0.73	0.70	0.28
AMP	0.23	−0.85	0.46	−0.01	0.63
AMX	0.62	−0.67	0.42	−0.49	0.30
SXT	0.75	−0.54	0.61	−0.40	−0.11
AMK	0.73	−0.07	0.60	−0.72	−0.50
GEN	0.73	0.20	0.53	−0.69	−0.76
CFM	0.16	−0.16	0.06	−0.28	0.10
MULTI	−0.14	−0.67	−0.16	−0.04	0.65
Semi-urban community	TVS	F−	Cl−	NO2−	PO43
Total FC	−0.42	0.66	0.07	0.09	0.48
AMP	0.12	−0.34	−0.19	0.31	−0.33
AMX	0.67	0.28	0.98	0.59	0.94
SXT	−0.20	0.63	0.29	0.29	0.64
AMK	0.91	−0.37	0.84	0.90	0.54
GEN	0.28	−0.95	−0.32	0.07	−0.73
CFM	−0.60	0.47	−0.27	−0.02	0.12
MULTI	−0.94	0.31	−0.83	−0.59	−0.46

).

3.3. Response to the WTP Type

As mentioned, the types of WTPs evaluated were mechanized (aerated lagoon, AL), where the hydraulic retention time is not more than 5 days, and non-mechanized (stabilization lagoon, SL), with a hydraulic retention time longer than 20 days.

This gives rise to different environments between them, which affect both the bacteria and the values of the physicochemical parameters found (Figure 4 and Table S3).

The efficiencies achieved by the WTP in the removal of FCs were 97% for the AL system and 81% for the SL system. The percent of antibiotic resistance was significantly different between the two systems (F = 20, p = 0.000018), also for antibiotics (F = 26, p ≤ 0.0000), where AMP, AMX, SXT and CFM were higher in the SL, with 67, 48, 2 and 25.8% versus the AL of 20, 13, 22 and 5.3%, respectively. GEN, MULTI and AMK were not significantly different, and the most effective drugs were GEN, AMK and their mixture, with 0% survival.

Pearson’s correlation allowed us to see the affinity that exists between the TVS and the survival of the FCs versus AMP, AMX, SXT and CFM, where the higher the organic content, the higher the survival rate (

Table 3. Pearson’s correlation between the physicochemical parameters and the percent survival of FCs when subjected to antibiotics (N = 14; p < 0.05). Those that were significant in the exit water of the WTP are underlined.

Aerated Lagoon	DO	Cl−	NO2−	NO3−
Total FC	−0.76	0.75	0.53	−0.67
AMP	−0.41	0.77	0.90	−0.21
AMX	−0.77	0.64	0.61	−0.66
SXT	0.59	−0.19	−0.15	0.35
AMK	−0.02	0.04	0.34	0.58
GEN	−0.79	0.40	0.48	−0.61
CFM	−0.82	0.37	0.48	−0.74
MULTI	0.11	0.37	0.33	0.00
Stabilization Lagoon	pH	EC	DO	ST	TVS	F−	NH3
Total FC	0.6	−0.63	0.6	−0.63	−0.63	−1.00	−0.95
AMP	−0.9	0.94	−0.9	0.94	0.94	0.36	0.00
AMX	−1.0	0.97	−1.0	0.97	0.97	0.46	0.11
SXT	0.0	−0.01	0.0	−0.01	−0.01	0.74	0.93
AMK	−1.0	1.00	−1.0	1.00	1.00	0.66	0.35
GEN	−0.3	0.32	−0.3	0.32	0.32	0.92	1.00
CFM	−0.6	0.58	−0.6	0.58	0.58	−0.22	−0.56
MULTI	0.4	−0.39	0.4	−0.39	−0.39	0.43	0.72

).

As can be observed, the solid contents correlate in a positive way, and oxygen in a negative way. That is, when the solid content is high and the dissolved oxygen is low, the resistance towards the antibiotic increases. This is reported by other authors [19], where they mention that factors such as pH, EC and DO affect the microbial community because they may impose stress conditions. Also, the solids present in the effluent indicate the removal efficiency of the WTP.

3.4. Evaluation of Multiresistance in Selected Strains

Selected strains from each sampling were isolated and identified with API 20E strips, and the following identification shown in

Table 4. Number of profiles and taxa found.

WTP	Sample	Numeral Profile	Significative Taxa	% ID
Aerated lagoon	AMP	334477357	Enterobacter sakazakii	93.6
	AMX	334477357	Enterobacter sakazakii	93.6
	SXT	504455257	E. coli 1	99.9
	AMK	334057357	Citrobacter koseri/farmeri	87.2
	GEN	704457357	E. coli 1	91.1
	CFM	330457357	Enterobacter cloacae	94.1
Stabilization lagoon	AMP	514455257	E. coli 1	99.9
	AMX	524477357	Serratia odorífera 1	50.4
	SXT	504455257	E. coli 1	99.9
	AMK	334057357	Citrobacter koseri/farmeri	87.2
	GEN	700457257	E. coli 1	85
	CFM	704457257	E. coli 1	99.9
	MULTI	104457257	E. coli 1	88.2

was achieved. The ID percentages are obtained from the API website based on the results obtained from the biochemical tests on the API strips. We were able to isolate one strain from the plates with every antibiotic. We state this to clarify that, despite reporting 0% of resistance towards the MULTI treatment on two occasions, we found very little colonies and isolated them.

These strains, being isolated from the samples where antibiotic resistance was evaluated, are known to be resistant to at least one antibiotic, but we wanted to evaluate whether those strains were resistant to other antibiotics using the antibiogram technique. These were ceftriaxone, cefuroxime, cefepime, ciprofloxacin, amoxicillin/clavulanate, penicillin, vancomycin, imipenem, linezolid, piperacillin/tazobactam, levofloxacin and ampicillin, assessed using a BBL Sensi-Disc™ (Becton, Dickinson, Sparks, MD, USA). We found that all the strains were resistant to those antibiotics. In an investigation carried out [16] on the comparison of resistance to the treatment of urinary infections, it was found that in the majority of antibiotics evaluated against samples of E. coli, the resistance rates exceeded 20%. These results suggest that the resistance towards one antibiotic could be accompanied by resistance to more antibiotics.

4. Discussion

It is a well-established fact that wastewater treatment reduces the amount of fecal coliform microorganisms. However, research has also shown that the proportion of resistant and multidrug-resistant bacteria increases in treated wastewater [5]. Many antibiotics are released into wastewater due to incomplete metabolism in humans or due to unused antibiotics. This results in the presence of antibiotic-resistant bacteria and antibiotic resistance genes in wastewater [4].

This study analyzed the water of an urban and a semi-urban population to determine the resistance of FCs to six of the most distributed antibiotics in the population. We took samples at the entrance and exit of the WTP to evaluate the effects of the type of treatment on the antibiotic resistance of the FC. As one can see in the graph, there are no significant differences in the percentage of resistance for most of the antibiotics between the communities the WTP serves. However, there are differences after the treatment, which clearly demonstrates that the type of WTP affects the percentage of microorganisms that show resistance to antibiotics.

The greatest resistance to antibiotics was definitively found in urban areas. Once the microorganisms pass through the depuration systems (Figure 3), their resistance to antibiotics increases. This is particularly evident in the FCs coming from the semi-urban area, where the SL depuration system allows for a stay time of 20 to 30 days. In comparison, the AL system used in the urban area treats domestic wastewater for only 5 days.

The aerated lagoon differs from the stabilization lagoon in two keyways. First, the aerated lagoon forms more sediment than the stabilization lagoon due to its longer water retention time. This is evident in Table 1, which shows that the stabilization lagoon has twice the TVS content of the aerated lagoon. Second, the Pearson correlations in Table 2 demonstrate a strong relationship between TVS and AMK.

This result clearly indicates that the type of WTP has a significant influence on the development of antibiotic resistance and the permanence and population increase of bacteria with the genes that support the antibiotics. The place of transfer of resistance genes is the sediment [8], so greater sediment levels and retention time will lead to a greater transfer of these genes and an increase in bacterial population size. This is evident in the two types of plants studied, where the stabilization lagoon has a higher solid content than the aerated lagoon. This results in a greater variety of FCs that are resistant to a greater number of antibiotics [20].

The significant correlations found clearly support the above-stated results. It was observed that the resistances were directly proportional to the presence of TVS (organic matter) for AMP, AMX and CFM (Table 3). In the semi-urban area (stabilization lagoon), the resistance increased. The resistance was up to four times greater than that in the original population in the semi-urban area (stabilization lagoon), in contrast to the urban area (Table 1 and Figure 1 and Figure 2), where the increases in resistance were not significant between the original FC population and that which exited the purification system (aerated lagoon).

AMP, AMX, and CFM showed a positive correlation between resistance and electrical conductivity (salt content). In contrast, for DO, temperature, and TS, these antibiotics demonstrated an inverse correlation. Manaia (2018) [19] asserts that a mesophilic or slightly high temperature, as well as a neutral pH, favors the development of antibiotic resistance. This is likely because these conditions allow for the proliferation of bacteria. Similarly, high levels of organic carbon (TVS) and low dissolved oxygen content encourage this phenomenon. Environmental stressors stimulate the resistance of microorganisms, which in turn increases resistance to antibiotics.

Bacteria develop resistance through the exchange of genetic information, including conjugation, transduction, and transformation. This horizontal transfer of genetic material allows them to become resistant to antibiotics and other substances [5]. The genes involved in resistance are located either in the bacterial chromosome or in plasmids, and can be transmitted in treatment systems with a long hydraulic retention time, such as stabilization lagoons, where they are under stress for long periods of time, which promotes these mechanisms [20]. This work observed an increase in resistance for some antibiotics of up to four times (from 17 to 67%).

It is crucial to acknowledge the alarming rise in antibiotic resistance, particularly given the significant reliance on treated domestic wastewater in agricultural activities. The potential risk to farmers is imminent, as they are in direct contact with pathogenic and opportunistic bacteria that are resistant to antibiotics. Furthermore, the distribution systems themselves can serve as a conduit for the spread of antibiotic resistance to opportunistic pathogens [9].

These studies must be used to determine the types of antibiotics that should be distributed in the population. This study demonstrated that the bacteria exhibited resistance to four antibiotics: AMP, AMX, SXT, and CFM. In lesser amounts, GEN demonstrated resistance, while AMK and multiresistant bacteria showed the least resistance to the antibiotics tested. In a comprehensive analysis of global reports from diverse community settings, the authors identified that the most prevalent antibiotic resistances in commensal E. coli strains were to oxytetracycline (78%), ampicillin (72%), tetracycline (67%), trimethoprim (67%), and others. A parallel investigation of hospital WTP effluent revealed that E. coli exhibited 74% resistance to ampicillin [21,22].

Other studies have definitively shown that clinically important bacteria such as Aeromonas and Pseudomonas display resistance to commonly used antibiotics [5]. Similarly, enterobacteria across the globe exhibit high resistance to ampicillin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, and nalidixic acid. The antibiogram analysis of the isolated strains confirmed these multi-resistances, demonstrating that they all exhibited resistance to other antibiotics.

Stressful environmental conditions for fecal microorganisms, such as the amount of dissolved oxygen, also influence this effect. Many resistance genes are associated with stressful environmental factors, so this is an important factor to consider. The results of this study clearly show that the number of microorganisms in treated wastewater is significantly reduced. However, it is important to note that most of these organisms have developed resistance to certain antibiotics. In the state of Durango, as well as in various parts of Mexico, treated wastewater from aeration facilities and stabilization lagoons is used for the irrigation of urban green areas and crops. Therefore, it is essential to monitor the presence and persistence of these microorganisms over time.