Next Article in Journal
Effect of K-Solubilizing Purple Nonsulfur Bacteria on Soil K Content, Plant K Uptake, and Yield of Hybrid Maize Grown on Alluvial Soil in a Dyke Area in Field Conditions
Previous Article in Journal
Cold-Resistant Lactic Acid Bacteria from Zamorano-Leonesa Donkey Milk: Isolation, Functional Screening, and Genome-Based Insights for Technological Applications
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Applied Microbiology
Volume 5
Issue 4
10.3390/applmicrobiol5040136
applmicrobiol-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Seasonal Variation in Bacterial Load and Genetic Diversity in Groundwater from Aïn Tawjdate, Morocco

by
Asmae Aboulkacem
1,
Hanane Zaki
1,
Amina Aboulkacem
2,
Tarik Ainane
1,*,
Rafail Isemin
3,
Fatouma Mohamed Abdoul-Latif
4 and
Ayoub Ainane
1,*
1
Superior School of Technology (EST-Khenifra), University of Sultan Moulay Slimane, P.O. Box 170, Khenifra 54000, Morocco
2
Innovation and Research Laboratory for the Improvement of Education and Training Professions, P.O. Box 242, Kenitra 14000, Morocco
3
Biocenter, Tambov State Technical University, 392000 Tambov, Russia
4
Medicinal Research Institute, Center for Research and Study of Djibouti, P.O. Box 486, Djibouti City 77101, Djibouti
*
Authors to whom correspondence should be addressed.
Appl. Microbiol. 2025, 5(4), 136; https://doi.org/10.3390/applmicrobiol5040136
Submission received: 20 October 2025 / Revised: 16 November 2025 / Accepted: 18 November 2025 / Published: 26 November 2025
Browse Figures
Versions Notes

Abstract

Groundwater represents an essential resource for domestic and agricultural use, and its physicochemical and microbiological quality directly affects public health. This study assessed the bacteriological quality of untreated well water in the province of Fez-Meknes, specifically in the Aïn Tawjdate area, and evaluated seasonal variations in bacterial contamination. During the spring and summer of 2023, groundwater samples were collected from several wells. A total of 139 bacterial strains were isolated and identified using API biochemical galleries. The most frequently detected species were Aeromonas hydrophila gr.1 (6.47%), Aeromonas hydrophila gr.2 (9.35%), Enterobacter cloacae (7.19%), Pseudomonas aeruginosa (10.07%), and Flavimonas oryzihabitans (6.47%), among others. Genetic variability among ten E. cloacae isolates was further explored using ERIC-PCR profiling; the strains differed by more than three fragments and showed less than 80% similarity; therefore, they were considered as distinct ERIC types. Statistical analyses (Chi-square, Fisher’s exact, Tukey HSD, one-way ANOVA, and two-sided Dunnett tests) revealed no significant differences in bacterial load between wells within the same season, with p-values > 0.05 according to ANOVA. However, a significant increase in contamination levels was observed in summer compared with spring. These findings highlight the potential health risks associated with the consumption of untreated groundwater and underline the need for regular microbiological monitoring and improved water treatment practices in rural communities.

1. Introduction

Groundwater is a vital resource for drinking water supply, agricultural irrigation, and many domestic activities [1]. In Morocco, its exploitation has expanded over the past several decades to meet population growth and growing water needs. Since the 1940s, the expansion of private irrigation based on pumping groundwater has intensified, particularly in the Saïss [2], Haouz [3], Souss [4] and Tafilalet oasis [5] regions. According to the National Water Strategy, groundwater represents approximately 20% of the country’s renewable resources, thus constituting a major pillar of socioeconomic and agricultural development [6]. However, this resource, once considered naturally protected from contamination, is now subject to multiple pressures linked to overexploitation, anthropogenic pollution, and climate change [7].
Morocco is currently experiencing a period of acute water stress, characterized by water availability of less than 1000 m3 per capita per year. Projections indicate a worsening of the situation, leading to a structural water shortage (less than 500 m3/person/year) by 2025 [8]. This scarcity increases dependence on groundwater, which is sometimes used without prior treatment for drinking, hygiene, or irrigation. However, the quality of this water is not always guaranteed. The use of contaminated water can lead to major health risks, including the transmission of waterborne diseases [9].
The quality of water intended for human consumption is based on physicochemical (pH, conductivity, turbidity, mineralization) and, above all, bacteriological parameters, which are direct indicators of possible fecal or environmental contamination [10]. The World Health Organization estimates that nearly 80% of infectious diseases worldwide are linked to poor water quality, inadequate sanitation conditions, or poor hygiene. Waterborne diseases, such as gastroenteritis, dysentery, and typhoid, still represent a considerable health burden in many rural areas where access to drinking water remains limited [11].
Pathogenic microorganisms present in contaminated water—bacteria, viruses, and protozoa—pose the main public health risks. Among them, opportunistic bacteria such as Pseudomonas aeruginosa, Enterobacter cloacae, and Aeromonas hydrophila are frequently detected in groundwater and can cause sometimes serious infections, particularly in immunocompromised individuals [12]. Furthermore, some studies have revealed that the groundwater in the city of Sebaa Ayoune (Fes-Meknes region) poses a serious health risk to the population. The spatio-temporal distribution of these microorganisms depends on numerous factors, such as the season, well depth, groundwater recharge conditions, and proximity to pollution sources (agricultural activities, sewer leaks, domestic waste) [13]. Their detection and identification are therefore an essential step in assessing the microbiological safety of water.
Given this issue, groundwater quality monitoring has become a public health priority. Several studies have highlighted the importance of regular monitoring to quickly detect any microbial contamination, assess seasonal variations, and guide preventive actions [14,15,16]. In regions with high agricultural activity, infiltration of fertilizers, wastewater, or contaminated runoff promotes the presence of pathogens in unprotected wells [17]. However, this water is often consumed directly by rural populations, sometimes in the absence of regular health monitoring [18].
The microbiological assessment of this water relies on the isolation and identification of bacteria using selective and non-selective media, followed by biochemical tests to determine the genera and species present [19]. API galleries, commonly used for the rapid identification of Enterobacteriaceae and other Gram-negative bacilli, constitute a reliable reference method for characterizing bacterial diversity [20]. Furthermore, statistical analyses—such as the χ2, Fisher, ANOVA or Tukey tests—allow bacterial loads to be compared between sites or seasons and the significance of the observed differences to be assessed [21].
In this context, the present study aims to evaluate the bacteriological quality of untreated groundwater collected in the Aïn Tawjdate region, Fez–Meknès province, and to determine the seasonal variability of contamination between spring and summer 2023. The specific objective is to isolate and identify the main bacterial species present using API galleries, to characterize the genetic variability of a subset of Enterobacter cloacae strains using ERIC sequences, and to statistically analyze the differences observed between the seasons and the wells studied. This work is part of an approach to prevent health risks associated with the consumption of untreated well water and contributes to a better understanding of the microbial dynamics of groundwater in a context of increasing water vulnerability in Morocco.

2. Materials and Methods

2.1. Study Area

This study was conducted in Ain Taoujdate, located in the Fez–Meknès Province of Morocco (33°56′00.7″ N; 5°12′51.1″ W). The area is characterized by intensive agricultural activity and a widespread reliance on groundwater for irrigation and domestic uses. Six operational wells that supply smallholder farms and peri-urban households were selected to represent typical hydro-agroecosystems in the commune. Selection criteria included routine abstraction for irrigation or household use, accessibility for repeated sampling, and landowner permission [22]. None of the wells applied in situ water treatment prior to collection. Geographic coordinates were recorded for each site, and a site sketch map was compiled to document land use, proximity to cropland and livestock holdings, and potential sanitary hazards. The regional context and well locations are shown in Figure 1.

2.2. Sampling Design and Field Procedures

Sampling was conducted during spring and summer of 2023, at a frequency of one collection every 15 days for each accessible well, following standard recommendations for microbiological monitoring of water. Across the study period, a total of 50 groundwater samples were collected: 20 samples during the dry, low-flow season and 30 samples during the rainy, flood-influenced season. Field logs documented the date and time of collection, well identification code, recent meteorological conditions, observed agricultural activities, and any anomalies that could influence microbial water quality. Before each grab, wells were purged to remove stagnant water from the riser or immediate bore by allowing continuous pumping or drawing for two to three minutes. All collections were performed aseptically. For each sampling event, 500 mL of groundwater was collected directly into pre-sterilized, screw-capped polypropylene bottles. Bottles were labeled with the well code, date and time, and sampler initials. No residual disinfectant was present in the water; therefore, sodium thiosulfate was not required. Samples were placed immediately in an insulated cooler with ice packs to maintain an approximate temperature of 4 °C during transport and were shielded from light to minimize microbial changes prior to processing. Transport to the laboratory was completed within six hours of collection. Upon laboratory receipt, samples were processed immediately. When multiple assays were needed on the same sample, subsampling was performed in a Class II biological safety cabinet to reduce cross-contamination [23].

2.3. Bacteriological Analyses: Overview and Rationale

Bacteriological analyses followed established procedures for water quality assessment and were aligned with Moroccan standards that are equivalent to international norms. Two analytical tracks were employed. The first focused on indicator organisms for sanitary quality: total coliforms, fecal (thermotolerant) coliforms, intestinal enterococci, and heterotrophic plate counts. The second targeted selected opportunistic or enteric bacteria of public health relevance to groundwater, including Pseudomonas aeruginosa, Aeromonas hydrophila, Salmonella spp., and Acinetobacter spp. The analytical framework combined membrane filtration with selective and nonselective culture media, followed by isolation and biochemical identification of representative colonies using standardized API identification galleries. Results for indicators are expressed as colony-forming units (CFU) per 100 mL for membrane filtration assays or CFU per mL for spread or deep-seeded plates. Presence/absence was used when appropriate for targeted organisms [24,25].

2.4. Membrane Filtration and Culture Conditions

Prior to filtration, sample bottles were mixed by gentle inversion to resuspend particulates. Depending on the target organism and prescribed method, 100 to 250 mL of water was filtered through sterile mixed-ester cellulose membranes with a nominal pore size of 0.22 µm and a diameter of 47 mm using a multi-place vacuum filtration unit. Filtration units were disinfected between samples by rinsing with 70% ethanol followed by flaming and air drying, and one sterile water blank was processed after every three environmental filtrations to verify procedural sterility. After filtration, membranes were transferred with flame-sterilized forceps onto the surface of the appropriate agar medium in 50 mm Petri dishes, grid side up, ensuring complete contact without air bubbles. Plates were inverted and incubated at the specified temperature and time for each method. Serial tenfold dilutions in sterile physiological saline (0.85% NaCl) were prepared when necessary to obtain countable plates (generally 20 to 200 colonies). Where turbid or highly contaminated samples produced too numerous to count (TNTC) plates, analyses were repeated at higher dilutions [26,27,28].

2.5. Indicator Organisms: Methods and Reporting

Indicator bacteria were enumerated using membrane filtration or plate count techniques aligned with Moroccan standards and their ISO counterparts [29]. Total and thermotolerant coliforms were enumerated by filtering 100 mL of water and placing membranes on Tergitol-7 agar, with incubation for 48 h at 44 °C for thermotolerant coliforms. Characteristic colonies were counted and expressed as CFU per 100 mL, adjusting for dilution. Intestinal enterococci were enumerated by filtering 100 mL and placing membranes on Slanetz and Bartley medium, incubated for 48 h at 37 °C; typical colonies were counted as CFU per 100 mL, with optional confirmation on bile-esculin azide agar. Heterotrophic bacteria were evaluated by deep seeding 1 mL of undiluted water into nutrient agar incubated at 22 °C for 72 h to obtain the heterotrophic plate count at 22 °C, reported as CFU per mL. Aerobic mesophilic bacteria were additionally assessed by plating appropriate aliquots onto Plate Count Agar (PCA) and incubating at 37 °C for 24 h, with results expressed as CFU per mL (Table 1).

2.6. Target Organisms: Selective Detection

Selected opportunistic and enteric bacteria were sought using established selective culture approaches. For Pseudomonas aeruginosa, 250 mL was filtered and the membrane was placed on Cetrimide agar, followed by incubation for 48 h at 37 °C; typical colonies with characteristic pigmentation and odor were recorded and subcultured for confirmation [33]. For Aeromonas hydrophila, 250 mL were filtered and the membrane was placed on RYAN base agar with incubation for 24 h at 37 °C; suspect colonies were purified for identification [34]. For Salmonella spp., 250 mL was filtered and the membrane was immersed in buffered peptone water supplemented with Tween 80 (2 g/L) for 24 h at 37 °C as pre-enrichment, followed by streaking onto selective agars as needed for presumptive identification prior to biochemical confirmation [35]. For Acinetobacter spp., 250 mL was filtered and membranes were placed on MacConkey agar with incubation for 24 h at 37 °C; non-lactose-fermenting colonies were selected and purified [36]. Aerobic mesophilic bacteria were assessed by filtering 250 mL and plating onto PCA with 24 h incubation at 37 °C when required to support quantification beyond the heterotrophic plate count at 22 °C (Table 2) [37].

2.7. Isolation, Purification, and Biochemical Identification

For each sample and culture condition, representative colonies displaying characteristic morphology were streak-purified on the corresponding nonselective agar until single-colony purity was achieved, generally within two to three passages. Pure isolates were examined by Gram staining and subjected to oxidase and catalase tests as preliminary screening. Species-level identification was performed using standardized API galleries according to the manufacturer’s instructions. The API 20E strip was used for Enterobacteriaceae and other Gram-negative rods, API 20NE for non-enteric Gram-negative rods, API Listeria where appropriate, and API Rapid strips for the rapid identification of clinical and environmental Gram-negatives [38]. Strips were inoculated with fresh suspensions in sterile saline to the required turbidity, and incubated at 37 °C for the recommended duration, and reactions were read and scored using the manufacturer’s database. Across the study, a total of 139 isolates were obtained from all wells and seasons combined; relative frequencies by genus and species were recorded for subsequent reporting (Table 3) [39].

2.8. Expression of Results and Data Management

For indicator organisms enumerated by membrane filtration, counts are expressed as CFU per 100 mL and were calculated as the number of characteristic colonies multiplied by the dilution factor and normalized to the filtered volume. For heterotrophic counts on nutrient agar at 22 °C and mesophilic counts on PCA at 37 °C, results are expressed as CFU per mL. Plates with fewer than 20 colonies were considered estimated and noted accordingly; plates with more than 200 colonies were recorded as TNTC and repeated at higher dilutions. For targeted organisms on selective media, results are reported as presence or absence at the sample level; when colony counts were feasible and countable, semi-quantitative results were recorded. All raw data, including plate photographs, colony counts, dilutions, and identification codes, were recorded on standardized laboratory worksheets and entered into an electronic database with audit trails to support reproducibility [40].

2.9. ERIC-PCR Typing of Enterobacter cloacae

ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR was performed to assess genetic variability among Enterobacter cloacae isolates. Ten isolates identified as E. cloacae by API 20E were selected for ERIC typing. Genomic DNA was prepared by thermal lysis. Briefly, isolates were grown overnight on nutrient agar at 37 °C. A loopful of cells was suspended in 200 µL of nuclease-free water, heated at 95 °C for 10 min, and centrifuged at 12,000× g for 5 min; the supernatant was used as template DNA. DNA concentration and purity were verified by spectrophotometry (A260/A280) and by electrophoresis on 1% agarose. PCRs were performed in a total volume of 25 µL containing 1.5 U of Taq DNA polymerase (Promega, Madison, WI, USA), 1× manufacturer-supplied buffer with 2.5 mM MgCl2, 50 µM of each dNTP, 10% (v/v) dimethyl sulfoxide, 1.7 mg/mL bovine serum albumin, 2 µM of each primer, and 5 µL of template DNA. The primers used were ERIC-2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) and ERIC-1R (5′-ATGTAAGCTCCTGGGGATTCAC-3′) (Table 4). Thermal cycling was carried out in a calibrated thermocycler with an initial denaturation at 95 °C for 7 min, and 35 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 2 min, followed by a final extension at 72 °C for 10 min. Negative controls without template and positive controls with a previously characterized E. cloacae isolate were included in each run. Aliquots of 10 µL of each PCR product were mixed with loading dye and subjected to electrophoresis on 1% agarose gels prepared in 1× TAE buffer. Gels were run at 90–100 V for 90–120 min with a 100 bp DNA ladder for sizing, stained with a nucleic acid dye, and visualized under UV or blue-light transillumination. Banding patterns were scored by two independent readers as binary presence/absence across defined molecular weight windows. Only reproducible bands observed in duplicate amplifications were retained. Profiles were summarized descriptively to document variability among the ten strains [41,42,43].

2.10. Quality Assurance and Biosafety

Quality assurance and quality control procedures were implemented throughout. Field blanks consisting of sterile water exposed at the sampling site were included once per field day. Filtration blanks were processed after every three environmental samples to detect laboratory or equipment contamination. Control strains appropriate for each selective medium were streaked periodically to verify medium performance and expected colony morphology. At least 10% of samples were analyzed in duplicate to assess analytical precision. Incubators were monitored daily with calibrated thermometers. Media lot numbers and expiration dates were recorded, and any batch that failed quality checks was discarded and replaced. All manipulations involving environmental water and potential pathogens were conducted under Biosafety Level 2 practices, with appropriate personal protective equipment, use of a biological safety cabinet for aerosol-generating steps, and autoclave decontamination of biological waste at 121 °C and 15 psi for 20 min [45].

2.11. Statistical Analysis

Statistical analyses were performed to evaluate spatial differences among wells and seasonal differences between spring and summer. Data were inspected for completeness and plausibility, and CFU counts were log10-transformed when necessary to approximate normality and stabilize variance [46]. Categorical outcomes such as presence or absence of specific taxa were compared using the Chi-square test; when expected cell counts were below five, Fisher’s exact test (two-tailed) was applied [47]. Continuous outcomes such as CFU counts were analyzed using one-way analysis of variance (ANOVA) to test for differences among wells within each season after verifying homoscedasticity with Levene’s test. Where ANOVA indicated significant differences, pairwise comparisons were conducted with Tukey’s Honestly Significant Difference (HSD) test [48]. Seasonal effects were assessed by comparing mean log10 CFU values between spring and summer. When multiple groups were compared against a common reference (e.g., comparing summer values to spring as the control condition), Dunnett’s two-sided test was used to control the family-wise error rate [49]. Nonparametric alternatives such as the Mann–Whitney U test were considered when normality or homoscedasticity assumptions were not met despite transformation. All tests were two-tailed with an alpha level set at 0.05. Analyses were performed using standard statistical software (e.g., SPSS version 22), and scripts, outputs, and data dictionaries were archived to ensure reproducibility.

2.12. Ethical and Administrative Considerations

Sampling was conducted on private properties with the consent of owners. No human or animal subjects were involved. To preserve privacy and protect infrastructure, reporting of well locations is limited to the commune level and anonymized well codes are used in all datasets and figures.

3. Results

The microbiological characteristics of groundwater samples collected from six wells in Ain Taoujdate (Morocco) in the spring and summer of 2023 are presented through four main analytical components, providing a comprehensive overview of the bacteriological profile of the study area. First, the enumeration of indicator bacteria, including total and fecal coliforms, intestinal enterococci, and heterotrophic microorganisms, was carried out to assess the sanitary quality of the water. Second, the isolation and identification of selected and opportunistic bacterial species were carried out using cultural and biochemical methods to characterize the diversity of culturable microorganisms present in the wells. Third, molecular analysis based on the Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) method was applied to a subset of Enterobacter cloacae isolates to assess genetic variability between strains. Finally, statistical analyses were conducted to compare bacterial abundance, diversity, and distribution between wells and between the two sampling seasons.

3.1. Indicator Bacteria

Indicator bacteria quantified by membrane filtration and plate count included total coliforms, fecal (thermotolerant) coliforms, intestinal enterococci, and heterotrophic bacteria (revivable microorganisms). Concentrations measured during summer were higher in magnitude than those recorded during spring for all four indicators. Table 5 summarizes the counts per 100 mL (for coliforms and enterococci) and per mL (for revivable microorganisms) by season.
Across the study, the measured total and fecal coliform concentrations exceeded the drinking-water reference value used in this work (10 CFU/100 mL) but remained below the irrigation-water reference value indicated (1000 CFU/100 mL) when expressed as total coliforms per 100 mL. Within each season, no significant differences in indicator counts among wells were detected by the statistical tests detailed below; between seasons, higher counts were recorded during summer sampling.

3.2. Cultivable Pathogenic and Opportunistic Bacteria

Culture-based isolation on selective and nonselective media followed by biochemical identification recovered a diverse set of bacterial taxa from the six wells. A total of 139 isolates were obtained across both seasons. The species lists per well and season are presented in Table 6. Species names are reported as identified by API galleries; where applicable, group designations reported at sampling are retained.
Across wells, Aeromonas hydrophila (both group 1 and group 2 designations), Enterobacter cloacae, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Flavimonas horyzihabitans were frequently recovered in one or both seasons. Several species were observed in only one season at specific wells, as indicated in Table 2. Qualitative presence/absence results for targeted organisms were recorded per sample, and the aggregated species lists per well and season are as shown.

3.3. Frequency of the Most Abundant Taxa

Proportions of the most frequently identified taxa among the 139 isolates are summarized in Table 6. Percentages are calculated as the number of isolates identified to that taxon divided by the total number of isolates recovered across both seasons. These five taxa collectively account for a substantial fraction of the isolates identified in this study. Remaining isolates were distributed among the other species listed in Figure 2.

3.4. ERIC-PCR Profiles of Enterobacter cloacae

ERIC-PCR typing was performed on ten Enterobacter cloacae isolates to assess banding profile variability (Figure 3). Amplification with primers ERIC-2 and ERIC-1R produced complex, multi-band patterns in the 200 bp to >1000 bp size range on 1% agarose gels. Band presence/absence was scored independently by two readers; only reproducible bands present in duplicate runs were retained for analysis. Dice similarity coefficients were computed from the binary matrices and used to construct an UPGMA dendrogram based on Pearson correlation. According to the applied criteria [50], banding pattern comparisons that differed by more than three fragments and showed <80% similarity were considered distinct ERIC types, whereas differences of one to three fragments with ≥80% similarity were considered ERIC subtypes. Based on the results obtained, we note that the percentage of similarity is between 20% and 80%, which suggests that the strains studied were considered as distinct ERIC types. The dendrogram revealed multiple ERIC profiles among the ten isolates, with clusters corresponding to higher similarity values and several singletons. Detailed similarity percentages and cluster assignments are provided in the accompanying gel image and dendrogram.
Analyses were conducted to evaluate associations between wells and detected species, to compare bacterial loads among wells within seasons, and to assess seasonal differences. Presence/absence of identified species across wells during spring was examined using Fisher’s exact test; no statistically significant association between wells and species detection was observed for spring samples. Chi-square testing across all wells and strains indicated no significant association between specific wells and particular strains, with p = 0.9 for the overall test. For quantitative comparisons among wells, one-way analysis of variance (ANOVA) was applied to log-transformed CFU counts (where appropriate) within each season. The ANOVA indicated no significant differences in mean bacterial counts among the six wells within season, with p-values > 0.05. Pairwise comparisons among wells using Tukey’s Honestly Significant Difference (HSD) test did not reveal significant differences; all pairwise p-values exceeded 0.6. Using Well 1 as the reference, Dunnett’s two-sided test comparing other wells to the reference yielded p-values > 0.4 for all comparisons. A two-way ANOVA considering well and bacterial strain type as factors was performed to explore potential interactions. For each well under study, p-values exceeded 0.5 for comparisons at the number or genus level, indicating no statistically significant differences attributable to the combination of well and strain factors within the analytical framework applied.

4. Discussion

The microbiological assessment of groundwater from the Ain Taoujdate region revealed clear evidence of bacterial contamination, highlighting potential risks for both environmental and public health. The observed concentrations of total and fecal coliforms, intestinal enterococci, and heterotrophic bacteria exceeded the limits established by the Moroccan and World Health Organization (WHO) standards for potable water, confirming that the sampled wells cannot be considered safe for direct human consumption [51]. These findings align with the notion that groundwater, even in rural or semi-urban regions, remains vulnerable to diffuse contamination from anthropogenic sources, including domestic wastewater infiltration, agricultural runoff, and inadequate well maintenance [52]. The consistent detection of coliforms and enterococci further supports their role as reliable indicators of organic and fecal pollution, respectively, as documented in previous works [53,54].
The elevated bacterial counts during the summer season, compared with spring, may be explained by several environmental and hydrological factors. High temperatures and enhanced evaporation during summer promote bacterial proliferation, while reduced dilution due to lower groundwater recharge favors higher microbial concentrations. Conversely, spring rainfall likely facilitates water renewal and dilution of contaminants. Similar seasonal variations have been reported in groundwater and surface water studies from North Africa and southern Europe, where microbial indicators tend to increase during warm and dry periods due to lower flow and higher nutrient availability [55].
The occurrence of potentially pathogenic and opportunistic species such as Pseudomonas aeruginosa, Aeromonas hydrophila, Enterobacter cloacae, and Flavimonas horyzihabitans is particularly concerning. These bacteria are known to cause infections in immunocompromised individuals and have been frequently isolated from contaminated aquatic environments. Pseudomonas aeruginosa, for instance, is recognized as a leading opportunistic pathogen capable of forming biofilms in water distribution systems and displaying intrinsic resistance to multiple antibiotics. The presence of Aeromonas hydrophila—a species associated with enterotoxins and cytotoxins—also suggests fecal or organic contamination, as previously noted in surface and groundwater studies conducted in North Africa. Similarly, Enterobacter cloacae is an environmental species that can become pathogenic under hospital or community settings, while Flavimonas horyzihabitans has been linked to rare but severe human infections. The repeated detection of these taxa across wells and seasons indicates their environmental persistence and ability to adapt to groundwater ecosystems, a phenomenon previously observed for environmental Pseudomonas and Acinetobacter spp. [56].
The diversity of the bacterial community, as revealed by API identification, suggests multiple sources of contamination. Wells surrounded by agricultural land may be influenced by livestock waste and manure infiltration, while peri-urban wells could receive domestic effluent contributions through defective septic systems. The structural integrity of well walls and the permeability of local soils appear to be determining factors in bacterial migration, consistent with prior observations that poorly constructed wells and permeable substrates facilitate the downward transport of microorganisms from surface to groundwater [57]. The detection of Vibrio, Providencia, and Proteus species in isolated cases supports the hypothesis of transient contamination events, possibly linked to episodic surface runoff or localized leakage.
ERIC-PCR typing of Enterobacter cloacae isolates provided molecular evidence of strain-level variability within the aquifer system. The multiple ERIC profiles identified among the ten tested isolates suggest the coexistence of genetically distinct lineages rather than a single clonal population, pointing toward multiple and possibly independent contamination inputs. The diversity of ERIC types with similarity values below 80% supports this interpretation and is consistent with Tenover criteria [58], which established that variations in more than three DNA fragments indicate distinct strain types. Such genetic heterogeneity has been reported in Enterobacter populations isolated from environmental matrices, reflecting their adaptability and the role of horizontal gene transfer in maintaining genetic diversity within contaminated ecosystems [59].
Statistical analyses confirmed the absence of significant differences in bacterial abundance and diversity among the six wells. The Chi-square and Fisher’s exact tests indicated no well-specific bacterial signature, suggesting that contamination sources are widespread and not limited to a single point. Similarly, one-way and two-way analyses of variance revealed no significant differences (p > 0.05) in bacterial counts or species composition among wells or between taxonomic groups, implying a uniform distribution of microbiological contamination across the study area. These results are in agreement with previous environmental microbiology studies reporting that microbial loads in shallow aquifers are often spatially homogeneous when contamination arises from diffuse or shared sources [60,61].
The comparison between spring and summer data, although not statistically significant at the 0.05 level, shows a clear trend toward higher bacterial loads during the dry season, consistent with the physicochemical and hydrological dynamics of semi-arid aquifers. Seasonal variations in temperature, water table depth, and infiltration rates may control microbial survival and transport. Warmer conditions typically favor bacterial growth and biofilm development, whereas increased precipitation and recharge events in spring promote dilution and partial flushing of microbial populations [62].
From a public health perspective, the detection of multiple opportunistic and potentially pathogenic species in untreated groundwater raises concern regarding the use of such water for domestic or agricultural purposes [63,64]. Although microbial counts remained below FAO guideline values for irrigation, their presence may still pose indirect risks through crop contamination and potential transmission to humans. Continuous, intermittent or seasonal pollution patterns should also be considered, as well as extreme and infrequent events, such as droughts and floods (WHO, 2022) [65]. Previous studies have demonstrated that irrigation with bacterially contaminated groundwater can facilitate the transfer of pathogens to vegetables and fruits, especially when overhead irrigation or short preharvest intervals are used [66,67]. The findings therefore underscore the necessity of systematic monitoring and periodic disinfection, particularly in rural areas lacking centralized water treatment infrastructure.
This study demonstrates that groundwater in Ain Taoujdate is bacteriologically contaminated, exhibiting seasonal variability and hosting diverse bacterial communities that include recognized opportunistic pathogens. Although concentrations comply with irrigation standards, the presence of fecal indicators and pathogenic strains suggests potential sanitary hazards. Continuous microbiological surveillance and improved well management practices are essential to safeguard groundwater quality. Future work should aim to elucidate contamination pathways and assess the potential transfer of resistance determinants within local microbial communities, contributing to more effective water safety strategies in semi-arid agricultural regions.

5. Conclusions

This study demonstrated that groundwater from the Ain Taoujdate region exhibits measurable bacteriological contamination, with seasonal variability and the recurrent presence of fecal indicator bacteria and opportunistic pathogens such as Pseudomonas aeruginosa, Aeromonas hydrophila, and Enterobacter cloacae. These findings confirm that untreated groundwater, while often used for irrigation and sometimes for domestic purposes, cannot be considered microbiologically safe without adequate monitoring and treatment. The results contribute to the growing body of evidence indicating that groundwater quality in agricultural zones is directly influenced by human activities, soil permeability, and well maintenance conditions. The detection of diverse bacterial species, some with pathogenic potential, underscores the need for systematic surveillance programs and stricter control measures in rural water management. Beyond local implications, this research highlights the broader importance of integrating microbiological monitoring into groundwater resource protection strategies, particularly in semi-arid regions where water scarcity drives reliance on untreated wells. Future work should combine molecular and physicochemical approaches to identify contamination sources, evaluate pathogen persistence, and assess potential antibiotic resistance. Sustained collaboration between scientific institutions and public health authorities will be important to prevent disease transmission and ensure the long-term sustainability and safety of groundwater resources.

Author Contributions

Conceptualization, T.A. and A.A. (Ayoub Ainane); Data curation, A.A. (Asmaa Aboulkacem); Formal analysis, A.A. (Asmaa Aboulkacem), H.Z., A.A. (Amina Aboulkacem) and F.M.A.-L.; Funding acquisition, T.A. and F.M.A.-L.; Investigation, F.M.A.-L.; Methodology, A.A. (Asmaa Aboulkacem), A.A. (Amina Aboulkacem) and F.M.A.-L.; Project administration, T.A.; Software, A.A. (Asmaa Aboulkacem), A.A. (Amina Aboulkacem), F.M.A.-L. and A.A. (Ayoub Ainane); Supervision, T.A. and A.A. (Ayoub Ainane); Validation, T.A. and A.A. (Ayoub Ainane); Visualization, H.Z., T.A. and R.I.; Writing—original draft, A.A. (Asmaa Aboulkacem), F.M.A.-L. and A.A. (Ayoub Ainane); Writing—review and editing, T.A., R.I., F.M.A.-L. and A.A. (Ayoub Ainane). All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in this study are included in the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Nahar, N.; Das, T.K.; Sadia, L.; Khatun, A.; Farzana, F.; Sarwar, S.; Shaibur, M.R. Hydrogeochemical evaluation and health implications of groundwater in Magura sadar upazila, Bangladesh: Application of GIS and Multivariate statistics. J. Chem. 2025, 2025, 8012445. [Google Scholar] [CrossRef]
  2. Ouafae, K.; Aouragh, M.H.; Hmaidi, A.E.; Ragragui, H.; Ouali, A.E. Integrated multi-index, GIS and multivariate statistical approaches for groundwater quality assessment in the Saïss Basin, Morocco. Euro-Mediterr. J. Environ. Integr. 2025, 10, 4217–4239. [Google Scholar] [CrossRef]
  3. Elgettafi, M.; Rochdane, S.; Elmandour, A.; Lorenzo, J.M.; Himi, M.; Casas, A. Br-Li and stable isotopes to induce groundwater salinity in crystalline and detrital aquifers: Oriental Haouz Morocco. J. Hydrol. Reg. Stud. 2025, 57, 102123. [Google Scholar] [CrossRef]
  4. Echogdali, F.Z.; Boutaleb, S.; Tariq, A.; Hamidi, M.; El Mekkaoui, M.; Ikirri, M.; Abdelrahman, K.; Uddin, G.; Akhtar, N.; Bendarma, A.; et al. Exploring groundwater patterns in Souss-Massa Mountainous Basin, Morocco: A fusion of fractal analysis and machine learning techniques on gravity data. J. Hydrol. Reg. Stud. 2024, 54, 101891. [Google Scholar] [CrossRef]
  5. Mouzoun, O.; Iharzi, C.; Kassou, A.; Essahlaoui, A.; Khrabcha, A. Lineament mapping using sig and optical and radar remote sensing: The case of the Tafilalet plain (Southeastern Morocco). In E3S Web of Conferences; EDP Sciences: Ulis, France, 2025; Volume 607, p. 02007. [Google Scholar]
  6. Mukherjee, A.; Jha, M.K.; Kim, K.W.; Pacheco, F.A. Groundwater resources: Challenges and future opportunities. Sci. Rep. 2024, 14, 28540. [Google Scholar] [CrossRef] [PubMed]
  7. Saxena, V. Water quality, air pollution, and climate change: Investigating the environmental impacts of industrialization and urbanization. Water Air Soil Pollut. 2025, 236, 73. [Google Scholar] [CrossRef]
  8. Dinar, A.; Tsur, Y. The Economics of Water Resources: A Comprehensive Approach; Cambridge University Press: Cambridge, UK, 2021. [Google Scholar]
  9. Marzouki, H.; Nordine, N.; Azzirgue, E.M.; Silva, J.C.E.D.; Cherif, E.K. From Contamination to Conservation: A Hydrochemical and Isotopic Evaluation of Groundwater Quality in the Semi-Arid Guire Basin (Morocco). Water 2025, 17, 1688. [Google Scholar] [CrossRef]
  10. Lora-Ariza, B.; Piña, A.; Donado, L.D. Assessment of groundwater quality for human consumption and its health risks in the Middle Magdalena Valley, Colombia. Sci. Rep. 2024, 14, 11346. [Google Scholar] [CrossRef]
  11. Liu, Q.; Deng, J.; Yan, W.; Qin, C.; Du, M.; Wang, Y.; Liu, J. Burden and trends of infectious disease mortality attributed to air pollution, unsafe water, sanitation, and hygiene, and non-optimal temperature globally and in different socio-demographic index regions. Glob. Health Res. Policy 2024, 9, 23. [Google Scholar] [CrossRef]
  12. Banda, B.V.; Mapoma, H.W.T.; Thole, B. Investigating Antibiotic Susceptibility of Pathogenic Micro-Organisms in Groundwater from Boreholes and Shallow Wells in T/A Makhwira, Chikwawa. Microbiol. Res. 2025, 16, 137. [Google Scholar] [CrossRef]
  13. Bousouis, A.; Bouabdli, A.; Ayach, M.; Lazar, H.; Ravung, L.; Valles, V.; Barbiero, L. Discrimination of Spatial and Temporal Variabilities in the Analysis of Groundwater Databases: Application to the Bourgogne-Franche-Comté Region, France. Water 2025, 17, 384. [Google Scholar] [CrossRef]
  14. Zhou, X.; Sun, J.; Yi, H.; Ye, T.; Zhao, Y.; Yang, Y.; Cui, J. Seasonal variations in groundwater chemistry and quality and associated health risks from domestic wells and crucial constraints in the Pearl River Delta. Environ. Sci. Process. Impacts 2025, 27, 936–949. [Google Scholar] [CrossRef]
  15. Bagordo, F.; Brigida, S.; Grassi, T.; Caputo, M.C.; Apollonio, F.; De Carlo, L.; Savino, A.F.; Triggiano, F.; Turturro, A.C.; De Donno, A.; et al. Factors influencing microbial contamination of groundwater: A systematic review of field-scale studies. Microorganisms 2024, 12, 913. [Google Scholar] [CrossRef]
  16. Gueroui, Y.; Bousbia, A.; Boudalia, S.; Touati, H.; Benaissa, M.; Maoui, A. Groundwater quality and hydrochemical characteristics in the upper Seybouse sub-basin, Northeast Algeria. Environ. Sci. Pollut. Res. 2024, 31, 26628–26645. [Google Scholar] [CrossRef]
  17. Petruța, N.L.; Sur, I.M.; Rusu, T.A.; Gabor, T.; Rusu, T. Integrated assessment of groundwater vulnerability and drinking water quality in rural wells: Case study from Ceanu Mare Commune, Northern Transylvanian Basin, Romania. Sustainability 2025, 17, 6530. [Google Scholar] [CrossRef]
  18. Ghosh, D.; Chaudhary, S.; Sarkar, S.; Singh, P. Water pollution in rural areas: Primary sources, associated health issues, and remedies. Water Resour. Manag. Rural. Dev. 2024, 15–28. [Google Scholar] [CrossRef]
  19. Vauloup, A.; Cébron, A. Development of a device to trap soil bacteria capable of degrading organic contaminants such as alkanes and polycyclic aromatic hydrocarbons. J. Hazard. Mater. 2025, 491, 137690. [Google Scholar] [CrossRef] [PubMed]
  20. Stobnicka-Kupiec, A.; Gołofit-Szymczak, M.; Cyprowski, M.; Górny, R.L. Monitoring of enteropathogenic gram-negative bacteria in wastewater treatment plants: A multimethod approach. Environ. Sci. Pollut. Res. 2024, 31, 37229–37244. [Google Scholar] [CrossRef]
  21. Petculescu, I.; Hynds, P.; Brown, R.S.; Boudou, M.; McDermott, K.; Majury, A. Development of a “big data” groundwater microbial contamination index and spatial comparisons with enteric infection rates in southern Ontario. Sci. Total Environ. 2024, 947, 174408. [Google Scholar] [CrossRef] [PubMed]
  22. Shuvo, R.M.; Chowdhury, R.R.; Chakroborty, S.; Das, A.; Kafy, A.A.; Altuwaijri, H.A.; Rahman, M.T. Geospatially Informed Water Pricing for Sustainability: A Mixed Methods Approach to the Increasing Block Tariff Model for Groundwater Management in Arid Regions of Northwest Bangladesh. Water 2024, 16, 3298. [Google Scholar] [CrossRef]
  23. Kim, K.H.; Kim, H.R.; Oh, J.; Choi, J.; Park, S.; Yun, S.T. Predicting leachate impact on groundwater using electrical conductivity and oxidation–reduction potential measurements: An empirical and theoretical approach. J. Hazard. Mater. 2024, 474, 134733. [Google Scholar] [CrossRef]
  24. Srivastava, S.; Kumar, A.; Gupta, R.; Malik, A. Geospatial mapping and cluster analysis of antibiotic-resistant Escherichia coli in drinking water of semi-arid areas. Environ. Monit. Assess. 2025, 197, 582. [Google Scholar] [CrossRef]
  25. Costa, I.C.; Siciliano, S.; Hauser-Davis, R.A.; Wosnick, N.; Rincon, G.; Roges, E.M.; Festivo, M.L.; Rodrigues, D.D.P.; Silva, J.L. Nunes An enteric microbiota assessment in sharks and rays from the Brazilian Amazon Coast. Environ. Biol. Fishes 2025, 108, 1909–1920. [Google Scholar] [CrossRef]
  26. Odewade, L.O.; Imam, A.A.; Adesakin, T.A.; Odewade, J.O. Assessment of human faecal contamination on groundwater quality and reporting consequent waterborne diseases in Funtua Metropolis, Katsina State, Nigeria. Front. Water 2025, 7, 1561777. [Google Scholar] [CrossRef]
  27. Yashir, N.; Sun, Q.; Zhang, X.; Ma, M.; Wang, D.; Feng, Y.; Song, X. Co-occurrence of microplastics, PFASs, antibiotics, and antibiotic resistance genes in groundwater and their composite impacts on indigenous microbial communities: A field study. Sci. Total Environ. 2025, 961, 178373. [Google Scholar] [CrossRef]
  28. Tunç, G.; Uçar, F.; Telli, O.; Lima, N.; Boiu-Sicuia, O.A. Fungal contaminants in Turkish bottled water and their mycotoxin-producing potential. GMS Hyg. Infect. Control. 2025, 20, Doc41. [Google Scholar] [PubMed]
  29. Debassi, B.; Allaoua, N.; Ghanem, N.; Hafid, H.; Benacherine, M.; Chenchouni, H. Assessment of water quality of groundwater, surface water, and wastewater using physicochemical parameters and microbiological indicators. Sci. Prog. 2025, 108, 00368504251348544. [Google Scholar] [CrossRef] [PubMed]
  30. ISO 9308-1:2014; Water Quality—Enumeration of Escherichia coli and Coliform Bacteria. Part 1: Membrane Filtration Method for Waters with Low Bacterial Background Flora. International Organization for Standardization (ISO): Geneva, Switzerland, 2014.
  31. ISO 7899-2:2000; Water Quality—Detection and Enumeration of Intestinal Enterococci. Part 2: Membrane Filtration Method. International Organization for Standardization (ISO): Geneva, Switzerland, 2000.
  32. ISO 6222:1999; Water Quality—Enumeration of Culturable Micro-Organisms—Colony Count by Inoculation in a Nutrient Agar Culture Medium. International Organization for Standardization (ISO): Geneva, Switzerland, 1999.
  33. Koujalagi, T.; Ruhal, R. Mitigating Health Risks Through Environmental Tracking of Pseudomonas aeruginosa. Curr. Microbiol. 2025, 82, 57. [Google Scholar] [CrossRef]
  34. Çaktü Güler, K.; Göktürk, I.; Yılmaz, F.; Araz, A.; Denizli, A. Plasmonic nanosensors for environmental pollutants sensing: Recent advances and perspectives. Essent. Chem. 2024, 1, 1–19. [Google Scholar] [CrossRef]
  35. Dong, Y.; Jiang, Z.; Hu, Y.; Jiang, Y.; Tong, L.; Yu, Y.; Wang, Y. Pathogen contamination of groundwater systems and health risks. Crit. Rev. Environ. Sci. Technol. 2024, 54, 267–289. [Google Scholar] [CrossRef]
  36. Feleni, U.; Morare, R.; Masunga, G.S.; Magwaza, N.; Saasa, V.; Madito, M.J.; Managa, M. Recent developments in waterborne pathogen detection technologies. Environ. Monit. Assess. 2025, 197, 233. [Google Scholar] [CrossRef]
  37. Álvarez, J.S.; Marciales, K.P.M. Microbiological identification of pathogens in water from educational centers in Norte de Santander. Rev. Cuid. 2025, 16, 2. [Google Scholar] [CrossRef]
  38. Chandler, L.J.; LaSala, P.R.; Whittier, S. Rapid devices and instruments for the identification of aerobic bacteria. In Manual of Commercial Methods in Clinical Microbiology, International ed., 2nd ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2016; pp. 21–55. [Google Scholar] [CrossRef]
  39. Ueno, A.; Sato, K.; Tamamura, S.; Murakami, T.; Inomata, H.; Tamazawa, S.; Igarashi, T. Gaoshiqia hydrogeniformans sp. nov., a novel hydrogen-producing bacterium isolated from a deep diatomaceous shale formation. Int. J. Syst. Evol. Microbiol. 2025, 75, 006802. [Google Scholar] [CrossRef]
  40. Pluym, T.; Waegenaar, F.; Dejaeger, K.; Dhoore, M.; Mestdagh, E.; Cornelissen, E.; De Gusseme, B. Membrane filtration reduces nutrient availability and invasion potential in drinking water systems, without affecting mature biofilms. Front. Microbiol. 2025, 16, 1622038. [Google Scholar] [CrossRef]
  41. Sraphet, S.; Tharasawatpipat, C.; Choo-In, S.; Kayee, P.; Namwong, S.; Budsabun, T.; Javadi, B. Comparative genomic analysis of cassava rhizospheric Bacillus subtilis using integrated in vitro and in Silico approaches with enterobacterial repetitive intergenic consensus (ERIC) sequences. Mol. Biol. Rep. 2025, 52, 489. [Google Scholar] [CrossRef]
  42. Louws, F.J.; Schneider, M.; de Bruijn, F.J. Assessing Genetic Diversity of Microbes Using Repetitive SequenceBased PCR (rep-PCR). In Environmental Applications of Nucleic Acid Amplification Technology; CRC Press: Boca Raton, FL, USA, 2024; pp. 63–94. [Google Scholar]
  43. Yang, Y.; Zhou, J.; Shi, D.; Yang, Z.; Zhou, S.; Yang, D.; Jin, M. Landscape of antibiotic resistance genes and bacterial communities in groundwater on the Tibetan Plateau, and distinguishing their difference with low-altitude counterparts. J. Hazard. Mater. 2023, 459, 132300. [Google Scholar] [CrossRef]
  44. Versalovic, J.; Koeuth, T.; Lupski, R. Distribution of repetitive DNA sequences in eubacteria and application to finerpriting of bacterial enomes. Nucleic Acids Res. 1991, 19, 6823–6831. [Google Scholar] [CrossRef]
  45. Blacksell, S.D.; Le, K.K.; Gleeson, L.J.; Stenos, J.; Graves, S.R.; Day, N.P.J. Assessing the evidence for effective biosafety risk management in Coxiella burnetii research. One Health 2025, 21, 101159. [Google Scholar] [CrossRef]
  46. Wybraniec, C.; Cournoyer, B.; Moussard, C.; Beaupère, M.; Lusurier, L.; Leriche, F.; Galia, W. Occurrence of 40 sanitary indicators in French digestates derived from different anaerobic digestion processes and raw organic wastes from agricultural and urban origin. Front. Microbiol. 2024, 15, 1346715. [Google Scholar] [CrossRef]
  47. Xu, R.; Li, J.; Li, X.; Song, W. Effect of coal mining subsidence on soil microbial communities in mining areas with high groundwater levels. Environ. Geochem. Health 2025, 47, 245. [Google Scholar] [CrossRef]
  48. Likando, N.M.; Chipandwe, M.S. Statistical investigation of climate and landfill age impacts on Kupferberg landfill leachate composition: One-way ANOVA analysis. Discov. Water 2024, 4, 40. [Google Scholar] [CrossRef]
  49. Liu, Q.; Zhou, H.; Sun, M.; Li, Q.; Fan, H.; Chen, H.; Yang, R. Improvement of soil structure and bacterial composition by long-term application of seaweed fertilizer. J. Soil Sci. Plant Nutr. 2023, 23, 5122–5132. [Google Scholar] [CrossRef]
  50. Gautam, H.; Maheshwari, B.; Mohapatra, S.; Sood, S.; Dhawan, B.; Kapil, A.; Tezpur, B. Clonal relationship among Acinetobacter baumannii isolates from different clinical specimens by ERIC-PCR. Int. J. Infect. Dis. 2022, 116, S18–S19. [Google Scholar] [CrossRef]
  51. El Baroudi, H.; Ouazzani, C.; Moustaghfir, A.; Er-Ramly, A.; Essebbahi, I.; El Baroudi, Y.; Balouch, L. Evaluation of drinking water quality and potential health risks on the population in Morocco. Desalin. Water Treat. 2024, 320, 100715. [Google Scholar] [CrossRef]
  52. Zhang, M.; Wang, L.; Zhao, Q.; Wang, J.; Sun, Y. Hydrogeochemical and anthropogenic controls on quality and quantitative source-specific risks of groundwater in a resource-based area with intensive industrial and agricultural activities. J. Clean. Prod. 2024, 440, 140911. [Google Scholar] [CrossRef]
  53. Aboulkacem, A.; Chahlaoui, A.; Soulaymani, A.; Rhazi-Filali, F.; Benali, D. Etude comparative de la qualité bactériologique des eaux des oueds Boufekrane et Ouislane à la traversée de la ville de Meknès (Maroc). Rev. Microbiol. Ind. San. Environ. 2007, 1, 10–22. [Google Scholar]
  54. Petculescu, I.; Hynds, P.; Brown, R.S.; Mcdermott, K.; Majury, A. An investigation of microbial groundwater contamination seasonality and extreme weather event interruptions using “big data”, time-series analyses, and unsupervised machine learning. Environ. Pollut. 2025, 368, 125790. [Google Scholar] [CrossRef]
  55. Silva, R.C.G.; Reinecke, R.; Copty, N.K.; Barry, D.A.; Heggy, E.; Labat, D.; Roggero, P.P.; Borchardt, D.; Rode, M.; Gómez-Hernández, J.J.; et al. Multi-decadal groundwater observations reveal surprisingly stable levels in southwestern Europe. Commun. Earth Environ. 2024, 5, 387. [Google Scholar] [CrossRef]
  56. Furtak, K.; Marzec-Grządziel, A. Microbial Hazard in Cultivated Soils Located in the Floodplains of the Vistula River Valley, Poland—Preliminary Research. Water Air Soil Pollut. 2025, 236, 614. [Google Scholar] [CrossRef]
  57. Janda, J.M. A group with emerging potential in the clinical and public health realms: The genus Providencia. Infect. Dis. 2025, 57, 699–725. [Google Scholar] [CrossRef]
  58. Tenover, F.C.; Arbeit, R.D.; Goering, R.V.; Mickelsen, P.A.; Murray, B.E.; Persing, D.H.; Swaminathan, B. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J. Clin. Microbiol. 1995, 33, 2233–2239. [Google Scholar] [CrossRef]
  59. Abraham, A.; Mtewa, A.G.; Chiutula, C.; Mvula, R.L.S.; Maluwa, A.; Eregno, F.E.; Njalam’mano, J. Prevalence of Antibiotic Resistance Bacteria in Manure, Soil, and Vegetables in Urban Blantyre, Malawi, from a Farm-to-Fork Perspective. Int. J. Environ. Res. Public Health 2025, 22, 1273. [Google Scholar] [CrossRef]
  60. Tyagi, S.; Sarma, K. Tracing the land use specific impacts on groundwater quality: A chemometric, information entropy WQI and health risk assessment study. Environ. Sci. Pollut. Res. 2024, 31, 30519–30542. [Google Scholar] [CrossRef]
  61. Çallı, K.Ö.; Chiogna, G.; Bittner, D.; Sivelle, V.; Labat, D.; Richieri, B.; Hartmann, A. Karst water resources in a changing world: Review of solute transport modeling approaches. Rev. Geophys. 2025, 63, e2023RG000811. [Google Scholar] [CrossRef]
  62. Waegenaar, F.; Pluym, T.; Vermeulen, E.; De Gusseme, B.; Boon, N. Impact of flushing procedures on drinking water biostability and invasion susceptibility in distribution systems. Appl. Environ. Microbiol. 2025, 91, e0068625. [Google Scholar] [CrossRef] [PubMed]
  63. Van Tuan, P.; Jiang, Y.; Stigter, T.; Zhou, Y. Understanding groundwater use and vulnerability of rural communities in the Mekong Delta: The case of Tra Vinh province, Vietnam. Groundw. Sustain. Dev. 2024, 25, 101095. [Google Scholar] [CrossRef]
  64. Šarc, A.; Kosel, J.; Stopar, D.; Oder, M.; Dular, M. Removal of bacteria Legionella pneumophila, Escherichia coli, and Bacillus subtilis by (super) cavitation. Ultrason. Sonochem. 2018, 42, 228–236. [Google Scholar] [CrossRef]
  65. World Health Organization (WHO). Guidelines for Drinking-Water Quality: Incorporating the First and Second Addenda; World Health Organization: Geneva, Switzerland, 2022. [Google Scholar]
  66. Rao, A.P.; Pradhan, A.K.; Patel, J. Persistence of foodborne bacterial pathogens on microgreens and soil irrigated with contaminated water. J. Food Prot. 2025, 88, 100594. [Google Scholar] [CrossRef]
  67. Christou, A.; Beretsou, V.G.; Iakovides, I.C.; Karaolia, P.; Michael, C.; Benmarhnia, T.; Chefetz, B.; Donner, E.; Gawlik, B.M.; Lee, Y.; et al. Sustainable wastewater reuse for agriculture. Nat. Rev. Earth Environ. 2024, 5, 504–521. [Google Scholar] [CrossRef]
Applmicrobiol 05 00136 g001
Figure 1. Location of the six monitored wells in Ain Taoujdate (Fez–Meknès, Morocco) and surrounding land use.
Figure 1. Location of the six monitored wells in Ain Taoujdate (Fez–Meknès, Morocco) and surrounding land use.
Applmicrobiol 05 00136 g001
Applmicrobiol 05 00136 g002
Figure 2. Most abundant bacterial taxa among all isolates.
Figure 2. Most abundant bacterial taxa among all isolates.
Applmicrobiol 05 00136 g002
Applmicrobiol 05 00136 g003
Figure 3. ERIC-PCR banding profiles for ten Enterobacter cloacae isolates from Ain Taoujdate wells, with corresponding UPGMA dendrogram based on Dice similarity.
Figure 3. ERIC-PCR banding profiles for ten Enterobacter cloacae isolates from Ain Taoujdate wells, with corresponding UPGMA dendrogram based on Dice similarity.
Applmicrobiol 05 00136 g003
Table 1. Bacteriological analysis methods for well water according to Moroccan standards (NM).
Table 1. Bacteriological analysis methods for well water according to Moroccan standards (NM).
Microorganisms SoughtAnalysis MethodCulture MediumIncubationStandard
Total coliforms and fecal coliformsFiltration of 100 mLTergitol-7 agar48 h at 44 °CNM 03.07.003 (ISO 9308-1) [30]
Intestinal enterococciFiltration of 100 mLSlanetz and Bartley48 h at 37 °CNM 03.7.006 (ISO 7899-2) [31]
Revivable microorganisms at 22 °CDeep seeding of 1 mLNutrient agar72 h at 22 °CNM 03.07.005 (ISO 6222) [32]
Table 2. Methods for detecting selected bacteria in well water.
Table 2. Methods for detecting selected bacteria in well water.
Microorganisms SoughtAmount of Water FilteredCulture MediumIncubation
Pseudomonas aeruginosa250 mLCetrimide48 h at 37 °C
Aeromonas hydrophila250 mLRYAN base agar24 h at 37 °C
Salmonella spp.250 mLBuffered peptone water + Tween 8024 h at 37 °C (pre-enrichment)
Acinetobacter spp.250 mLMacConkey24 h at 37 °C
Aerobic mesophilic bacteria250 mLPlate Count Agar (PCA)24 h at 37 °C
Table 3. Different kinds of API strips used in this study.
Table 3. Different kinds of API strips used in this study.
API GalleryStrainsReference
20EEnterobacteriaceae and other Gram-negative rodsGalerie d’identification API-Diagnostic Clinique, bioMérieux France.
20NEnon-enteric Gram-negative rodsGalerie d’identification API-Diagnostic Clinique, bioMérieux France.
API Rapid 20ERapid identification of Enterobacteriaceae in 4 h.Galerie d’identification API-Diagnostic Clinique, bioMérieux France.
API ListeriaListeriaGalerie d’identification API-Diagnostic Clinique, bioMérieux France.
Table 4. ERIC primers used for Enterobacter cloacae typing.
Table 4. ERIC primers used for Enterobacter cloacae typing.
TargetPrimerSequence (5′ → 3′)Fragment Size (bp)Reference
ERIC lociERIC-2AAGTAAGTGACTGGGGTGAGCGVariableVersalovic et al., 1991 [44]
ERIC lociERIC-1RATGTAAGCTCCTGGGGATTCACVariableVersalovic et al., 1991 [44]
Table 5. Indicator bacteria counts by season.
Table 5. Indicator bacteria counts by season.
Microorganisms SoughtUnitSpring (Maximum Reported)Summer (Maximum Reported)
Total coliformsCFU/100 mL158303
Intestinal enterococciCFU/100 mL80103
Fecal coliformsCFU/100 mL90205
Revivable microorganisms (at 37 °C)CFU/mL145276
Table 6. Bacterial species isolated by well and season.
Table 6. Bacterial species isolated by well and season.
WellSpring (Species Identified)Summer (Species Identified)
Well 1Acinetobacter calcoaceticus; Aeromonas hydrophila gr.1; Aeromonas hydrophila gr.2; Citrobacter freundii; Comamonas testosteroniAcinetobacter calcoaceticus; Aeromonas hydrophila gr.1; Aeromonas hydrophila gr.2; Citrobacter freundii; Enterobacter amnigenus 2; Serratia odorifera
Well 2Aeromonas hydrophila gr.1; Aeromonas hydrophila gr.2; Enterobacter cloacae; Escherichia coli (type 1); Pseudomonas aeruginosa; Flavimonas horyzihabitans; Pseudomonas fluorescens; Citrobacter freundii; Comamonas testosteroni; Enterobacter amnigenus 2; Klebsiella ornithinolytica; Photobacterium damsela; Serratia liquefaciens; Stenotrophomonas maltophilia; Pseudomonas alcaligenesAeromonas hydrophila gr.1; Enterobacter cloacae; Pseudomonas aeruginosa; Flavimonas horyzihabitans; Pseudomonas fluorescens; Citrobacter freundii; Comamonas testosteroni; Klebsiella ornithinolytica; Photobacterium damsela; Serratia liquefaciens; Stenotrophomonas maltophilia; Hafnia alvei; Klebsiella oxytoca; Pasteurella pneumotropica; Pseudomonas stutzeri
Well 3Acinetobacter calcoaceticus; Escherichia coli (type 1); Pseudomonas fluorescens; Citrobacter freundii; Comamonas testosteroni; Proteus mirabilisAcinetobacter calcoaceticus; Aeromonas hydrophila gr.1; Aeromonas hydrophila gr.2; Enterobacter cloacae; Escherichia coli (type 1); Pseudomonas aeruginosa; Flavimonas horyzihabitans; Pseudomonas fluorescens; Comamonas testosteroni; Enterobacter amnigenus 2; Klebsiella ornithinolytica; Photobacterium damsela; Stenotrophomonas maltophilia; Acinetobacter cloacae; Ochrobactrum anthropi
Well 4Aeromonas hydrophila gr.1; Aeromonas hydrophila gr.2; Pseudomonas aeruginosa; Flavimonas horyzihabitans; Pseudomonas fluorescens; Enterobacter amnigenus 2; Klebsiella ornithinolytica; Photobacterium damsela; Serratia liquefaciens; Stenotrophomonas maltophilia; Acinetobacter baumannii; Enterobacter sakazakii; Providencia rettgeri; Pseudomonas horyzihabitansAeromonas hydrophila gr.1; Aeromonas hydrophila gr.2; Enterobacter cloacae; Escherichia coli (type 1); Flavimonas horyzihabitans; Enterobacter amnigenus 2; Pseudomonas fluorescens; Citrobacter freundii; Comamonas testosteroni; Klebsiella ornithinolytica; Photobacterium damsela; Serratia liquefaciens; Stenotrophomonas maltophilia; Aeromonas salmonicida; Enterobacter aerogenes; Raoultella ornithinolytica; Serratia marcescens
Well 5Acinetobacter calcoaceticus; Escherichia coli (type 1); Citrobacter freundii; Serratia liquefaciens; Stenotrophomonas maltophiliaAcinetobacter calcoaceticus; Aeromonas hydrophila gr.1; Escherichia coli (type 1); Pseudomonas aeruginosa; Citrobacter freundii; Pantoea spp. 4; Pseudomonas luteola; Vibrio parahaemolyticus
Well 6Aeromonas hydrophila gr.2; Pseudomonas aeruginosa; Bordetella sp.; Vibrio hollisaeAeromonas hydrophila gr.1; Aeromonas hydrophila gr.2; Pseudomonas fluorescens; Comamonas testosteroni; Klebsiella ornithinolytica; Eikenella corrodens
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Aboulkacem, A.; Zaki, H.; Aboulkacem, A.; Ainane, T.; Isemin, R.; Mohamed Abdoul-Latif, F.; Ainane, A. Seasonal Variation in Bacterial Load and Genetic Diversity in Groundwater from Aïn Tawjdate, Morocco. Appl. Microbiol. 2025, 5, 136. https://doi.org/10.3390/applmicrobiol5040136

AMA Style

Aboulkacem A, Zaki H, Aboulkacem A, Ainane T, Isemin R, Mohamed Abdoul-Latif F, Ainane A. Seasonal Variation in Bacterial Load and Genetic Diversity in Groundwater from Aïn Tawjdate, Morocco. Applied Microbiology. 2025; 5(4):136. https://doi.org/10.3390/applmicrobiol5040136

Chicago/Turabian Style

Aboulkacem, Asmae, Hanane Zaki, Amina Aboulkacem, Tarik Ainane, Rafail Isemin, Fatouma Mohamed Abdoul-Latif, and Ayoub Ainane. 2025. "Seasonal Variation in Bacterial Load and Genetic Diversity in Groundwater from Aïn Tawjdate, Morocco" Applied Microbiology 5, no. 4: 136. https://doi.org/10.3390/applmicrobiol5040136

APA Style

Aboulkacem, A., Zaki, H., Aboulkacem, A., Ainane, T., Isemin, R., Mohamed Abdoul-Latif, F., & Ainane, A. (2025). Seasonal Variation in Bacterial Load and Genetic Diversity in Groundwater from Aïn Tawjdate, Morocco. Applied Microbiology, 5(4), 136. https://doi.org/10.3390/applmicrobiol5040136

Article Metrics

Article metric data becomes available approximately 24 hours after publication online.
Back to TopTop