This study is part of an ongoing hospital-based diarrhea surveillance program across three health facilities in Puri that is aimed at detecting diarrhea-causing etiological agents in hospitalized patients with acute diarrhea. This study particularly focused on the detection of ETEC and their antibiogram.

The present study has been ongoing since 2011, following the administration of Shanchol—a bivalent oral cholera vaccine (OCV)—to a cholera-vulnerable population in the Satyabadi Block of Puri district. The objective was to evaluate the vaccine’s efficacy by administering two doses through the existing public health infrastructure, following the first round of clinical trials conducted in Kolkata (Kar et al., 2014). This surveillance study was conducted in the following three hospitals in 2022: Infectious Disease Hospital (IDH), Puri; Pediatric Hospital, Puri; and Area Hospital in Satyabadi Block, Puri. Due to its religious significance, Puri receives thousands of pilgrims every day from across the country and around the world. The resulting overcrowding creates unhygienic conditions, leading to the emergence of diarrhea, particularly during the rainy season. Puri is surrounded by numerous rural villages to the northeast and northwest, with a catchment population of more than 150,000. Both local inhabitants and tourists in Puri constitute the vulnerable population at risk for diarrhea.

In Odisha, each year, higher rates of diarrhea are expected from the month of June to the month of November, corresponding with the pre-monsoon, monsoon, and immediate post-monsoon periods. These months are marked by elevated temperatures, heavy rainfall, and flooding, which contribute to unsafe water and poor sanitation, thereby increasing the risk of diarrheal diseases. Considering this period as the high-diarrhea season, our study was designed to collect rectal swab samples once a week from the three health facilities in Puri District.

According to the WHO guidelines, diarrhea was defined as the passage of three or more loose or liquid stools within any 24-h period during the 3 days before presentation or the passage of one or two loose/liquid stools accompanied by any signs of dehydration (World Health Organization, 2005). Patients with chronic gastrointestinal disorders (e.g., Crohn’s disease, ulcerative colitis) that cause recurrent diarrhea and those who had recently used antibiotics were excluded. From May to November 2022, we enrolled hospitalized patients who presented with acute diarrhea. During the investigation, non-randomly selected rectal swab samples were collected from the hospitalized patients with acute watery diarrhea, prior to the administration of antibiotics, and stored in Cary–Blair transport (CBT) medium. Due to administrative constraints and limited manpower, samples were collected once a week during routine hospital visits by staff from the Regional Medical Research Centre (RMRC) who traveled from Bhubaneswar (approximately 60 km away). As a result, non-random sampling was adopted to maximize patient enrolment during these scheduled visits, especially in the challenging post-COVID period. Demographic data were collected using pre-structured forms, with the consent of each patient or attendant. The patient’s dehydration status—categorized as none, mild, moderate, or severe—was examined and assessed by a physician based on clinical signs, as described by the WHO. A pre-structured questionnaire covering demographic background, medical history, and previous treatment was completed by the patient or their attendant.

Immediately after collection, the rectal swab samples in CBT were transported to the Indian Council of Medical Research (ICMR)-RMRC Microbiology Laboratory for processing. The rectal swabs were inoculated onto thiosulphate–citrate–bile salts–sucrose (TCBS) agar, MacConkey agar, and Hektoen enteric agar, followed by enrichment in Alkaline Peptone Water (APW) to isolate V. cholerae and other major diarrhea-causing pathogens. Typical colonies were selected and tested biochemically to identify major diarrhea-causing bacterial pathogens using conventional methods.

At least three typical pink colonies on MacConkey agar were selected per sample for DNA extraction using the boiling method, as described in a previous study (Dutta et al., 2013). E. coli strains were confirmed genetically by detecting the alr marker gene using a monoplex PCR assay (Yokoigawa et al., 1999). The E. coli strains that tested positive for the alr gene were subjected to a multiplex PCR assay for the detection of various virulent genes for the identification of DEC. The multiplex PCR assay was conducted to detect DEC pathotypes: ETEC (elt and est), EPEC (eae and bfpA), and EAEC (aatA), using specific primer pairs described previously (Panchalingam et al., 2012). PCR amplification was performed using the appropriate volumes of 10x amplification buffer (500 mM KCL, 100 mM Tris HCL, 15 mM MgCl2, PH-8.3), 2.5 mM of each deoxynucleoside triphosphate, 10Pmole of each primer, 1.25 units of Taq DNA polymerase (Mumbai, India: HiMedia Laboratories, LLC), and 5 μL of template DNA. The reaction volume was adjusted to 25 μL using sterile triple-distilled water. The PCR assay was performed in an automated thermal cycler (Mumbai, India: HiMedia Laboratories, LLC) for 30 cycles using conditions described previously (Dutta et al., 2013). Aliquots of the PCR product were analyzed using agarose gel electrophoresis (1.8% wt/vol) in Tris/Borate/EDTA buffer, stained with ethidium bromide, and visualized under UV illumination. Positive controls for ETEC, EPEC, and EHEC and a negative control, Salmonella enterica serovar Typhi (Provided by Dr. Asish Kumar Mukhopadhyay, the National Institute of Cholera and Enteric Diseases (NICED), Kolkata), were included in each PCR assay set. The PCR method was initially standardized at the NICED, Kolkata, using specific primers targeting the virulence genes of toxigenic E. coli strains.

Antimicrobial susceptibility testing of the isolated DEC was performed on Mueller–Hinton agar using the Kirby–Bauer disc diffusion method [the Clinical and Laboratory Standards Institute (CLSI), Wayne, PA, 2006] with a panel of antibiotic discs: tetracycline (T 30 μg), gentamicin (GM,10 μg), streptomycin (S 10 μg), chloramphenicol (C, 30 μg), amikacin (AK, 30 μg), norfloxacin (NX 10 μg), neomycin (N, 30 UI), ampicillin (AM, 10 μg), ciprofloxacin (CP, 5 μg), ofloxacin (OF, 5 μg), furazolidone (FZ, 100 μg), azithromycin (ATZ, 15 μg), and doxycycline (DO 30 μg). According to the Clinical and Laboratory Standards Institute (CLSI) guidelines (Albert et al., 1995), susceptibility was categorized as sensitive or resistant based on the diameter of the zone of inhibition. Toxigenic E. coli positive control strains were provided as a gift by Dr. Ashish Mukhopadhya from the National Institute of Cholera and Enteric Diseases (NICED), Kolkata.

Data analysis was conducted using the software SPSS (version 27), R (version 4.3.1), and Python (version 3.10). Descriptive statistics, including means, standard deviations, and frequencies, were used to summarize the data. For inferential analysis, specific tests such as independent t-tests, a one-way ANOVA, and the chi-squared tests were employed to assess differences between the groups. The assumptions of normality and homogeneity of variance were evaluated using the Shapiro–Wilk test and Levene’s test, respectively. When these assumptions were violated, non-parametric alternatives, such as the Mann–Whitney U test and the Kruskal–Wallis test, were applied. Correlations were assessed using either Pearson’s or Spearman’s correlation, depending on the data distribution. A regression analysis was also performed, where appropriate, to examine predictive relationships. Statistical significance was set at a p-value of < 0.05. The correlation between ETEC prevalence and age was assessed using Pearson’s correlation to determine the strength and direction of the relationship. Pearson’s correlation was implemented as it measures the linear association between two continuous variables. The analysis was performed using SPSS, R, and/or Python, and statistical significance was set at a p-value of < 0.05. The correlation coefficient (R-value) and p-value were calculated to interpret the relationship.

The ethical committee of RMRC, Bhubaneswar approved the study. Written informed consent was obtained from each patient before the collection of samples. Guardians provided written consent in cases of children.

A bacteriological analysis of the 256 rectal swabs revealed the following pathogens: E. coli spp. (172, 67.2%), Shigella spp. (12, 4.7%), Salmonella spp. (four, 1.7%) but no V. cholerae isolates. Of the total 172 presumptive E coli spp., 108 (62.8%) were genetically confirmed as E. coli through the detection of the alr gene. Of the 108 genetically confirmed E. coli spp., 72 (66.7%) were identified as DEC. Overall, 29% (72/256) of the total patients were DEC-positive, confirmed by the detection of different virulent genes in E. coli strains. Among the total samples, ETEC strains (20.3%, 52/256) were identified as the predominant pathogen, followed by EAEC (6.3%, 16/256) and EPEC (2.3%, 6/256) (Figure 1). Of the total DEC-positive cases, ETEC accounted for the highest proportion, 70.3% (52/72), as the predominant diarrhea-causing pathogen. Genetic screening did not identify any strains belonging to EHEC. Combinations of infections were detected among patients with diarrhea, with the following combinations: ETEC/EPEC 0.8% (2/256), ETEC/EAEC 2.3% (6/256), and EPEC/EAEC 0.8% (2/256) (Figure 1).

The statistical analysis revealed a significant association between the specific DEC strains and the age groups of patients with diarrhea (Table 1). ETEC was significantly more prevalent among children aged 0–23 months compared to those aged ≥15 years (chi-squared = 36.70, p < 0.00001). The odds of ETEC infection among children aged 0–23 months were 8.56 times higher than those in the ≥15-year group (95% CI: 2.77–26.43). Both EPEC and EAEC exhibited significant age-related differences (chi-squared = 14.19, p = 0.003 for EPEC; chi-squared = 19.14, p = 0.00026 for EAEC). However, these strains were less prevalent in the younger age groups compared to the older patients, with odds ratios of 0.07 (95% CI: 0.004–1.28) and 0.24 (95% CI: 0.07–0.86) for EPEC and EAEC, respectively. Mixed infections (e.g., ETEC/EPEC and EPEC/EAEC) did not demonstrate significant associations with the age groups, potentially due to smaller sample sizes. A negligible negative correlation was observed between age and ETEC prevalence (R = −0.013), indicating that ETEC occurrence did not substantially vary across the age groups. This finding suggests that ETEC prevalence does not significantly change with increasing age.

Among the different types of DEC, the potential pathotype was determined based on the higher prevalence of virulent genes, either individually or in combination with other genes (Table 2). In the present study, the prevalence of est was 14.8% (38/256), followed by elt at 11.7% (30/256) and a mix of elt/est at 6.2% (16/256). Overall, ETEC (20.3%, 52/256), harboring elt, est, and elt/est, was identified as the dominant pathotype strain-causing diarrhea. In the case of EPEC, only 2.3% of the samples (6/256) amplified eae, and no bfpA, either alone or in combination with eae, was detected. This finding indicates that the patients in our study were infected with atypical EPEC strains, while no typical EPEC strains were prevalent. Among the EAEC isolates (n = 16), all harbored the aatA gene, representing 6.25% (16/256) of total samples that tested positive for aatA.

Among the different DEC pathotypes, ETEC showed the highest prevalence at 20.3% (52/256) in our study. Age-specific distribution revealed that ETEC was detected in children under 5 years and adults over 15 years, with no isolates of ETEC observed in the 5–15-year age group—possibly due to the smaller number of samples or natural immunity within that age range (Table 2). Children under 5 years accounted for 9.4% (24/256) of the ETEC cases compared to 11% (28/256) in the adults. The difference was minimal and not statistically significant. Overall, the prevalence of ST-ETEC (14.8%, 38/256) was higher compared to LT-ETEC (11.8%, 30/256) across all age groups. Age-specific distribution of ST- and LT-ETEC showed that LT-ETEC was more prevalent in adults (8.6%, 22/256) compared to children under 5 years (3%, 8/256). ST-ETEC was detected in 7.8% (20/256) of children and 7% (18/256) of adults, indicating a negligible difference. Among adults, LT-ETEC (8.6%) was slightly more common than ST-ETEC (7%), although the difference was small. In contrast, among children under 5 years, ST-ETEC (7.8%, 20/256) was more prevalent than LT-ETEC (3%, 8/256). A higher prevalence of ETEC infection was observed in male participants (75%) compared to female participants (25%). Of the total ETEC enterotoxin-positive cases, ST accounted for 45.3%, LT for 35.7%, and the combination of ST/LT for 19% (Table 2). A higher proportion (58.8%, 40/68) of single enterotoxin-producing strains—either ST or LT—was observed in adults (>15 years). Among adults, the prevalence of LT (42.4%) was higher than that of ST (34.6%); however, among children under 5 years, the prevalence of ST (29.4%) was higher than that of LT (11.7%) (Table 3).

The observation of clinical symptoms in the ETEC-infected children and adults with acute diarrhea revealed that watery stool was the most common symptom among almost all children and adults (Table 4). It was observed that a higher percentage of children experienced vomiting (81%) and fever (14%) compared to the adults, who exhibited vomiting in approximately 75% of cases and fever in 6%. Upon admission, a higher proportion of ETEC-infected adults exhibited severe (36%) and moderate (45%) levels of dehydration compared to the pediatric patients. Intravenous (IV) rehydration therapy was required in 54% of the adults compared to 13% of the children, while the remaining cases in both groups were managed with oral rehydration. Approximately 50% of the ETEC-infected patients experienced abdominal pain.

Diarrhea caused by ETEC was observed from May to October 2022. The month-wise distribution of ETEC strains in the samples was as follows: 14.8% (4/27) in May, 44.3% (27/61) in June, 26.7% (16/60) in July, 4.2% (2/47) in August, 6.4% (2/31) in September, 4.5% (1/22) in October, and 0% (0/8) in November 2022 (Figure 2). Isolation of ETEC began in the pre-monsoon month of May (14.8%), peaked during the monsoon in the month of June (44.3%), and subsequently declined in the following months, with no ETEC isolated in November. Increased levels of ST-ETEC and lower levels of LT-ETEC were observed throughout the study period. The highest combination of LT + ST was detected during the peak summer month of June. Concurrently, as per the Meteorological Department of Bhubaneswar, Odisha, the highest environmental temperatures recorded were 38° C, 39° C, 36° C, and 37° C in May, June, July, and August 2022, respectively. The temperature gradually declined in the subsequent months. The amplification of the est gene was higher in June (n = 13) and July (n = 10) than in May (n = 1) and August (n = 1).

The antibiotic resistance pattern is shown in Figure 3. The majority of the ETEC strains (95%) were multidrug-resistant (MDR) (resistant to three or more groups of antibiotics). The ETEC antibiotic resistance profile was as follows: tetracycline (TE, 52%), ampicillin (AMP, 85%), neomycin (N, 67%), furazolidone (FR, 57%), norfloxacin (Nx, 85%), ciprofloxacin (CIP, 67%), ofloxacin (OF, 76%), azithromycin (AZM, 85%), and doxycycline (DO, 67%). More than 50% of the ETEC strains exhibited a combination of multiple drug resistance profiles, including AMP, NX, OF, DO, FR, TE, AZM, CIP, and NE. In contrast, the strains were susceptible to S, C, GEN, and AK.

ETEC is a well-established leading cause of diarrhea in infants, particularly affecting children aged 0–23 months. However, despite this peak in infancy, the weak negative overall correlation (R = −0.013) indicated that the prevalence of ETEC did not consistently increase or decrease with age across the entire sample. While the present data indicate a relatively uniform distribution, it is critical to consider additional risk factors, such as hygiene practices, dietary habits, and environmental exposure, that may influence ETEC transmission beyond age alone. Furthermore, the limited statistical significance observed in mixed infections could be attributed to sample size constraints, highlighting the need for large-scale studies to gain deeper insights.

Notably, this study reports ETEC as the predominant enteric pathogen causing diarrhea in 2022, replacing V. cholerae, which has historically been the leading cause of diarrhea in this region. Similar trends have been observed in other cholera-endemic regions, where fluctuations in enteric pathogen prevalence occur due to environmental and epidemiological factors. The present findings suggest that both children under 5 years of age and adults aged 20–60 years are at the highest risk for ETEC-associated diarrhea, which is consistent with previous studies (Chakraborty et al., 2024; Chandra et al., 2012). Global studies have also highlighted significant geographical variation in ETEC prevalence, with reported rates ranging from 18 to 38% among symptomatic children in countries such as Bangladesh, Mexico, Peru, Egypt, Argentina, Nicaragua, and Tunisia (Rivera et al., 2010; Hien et al., 2008; Bueris et al., 2007). The present study reports an overall ETEC prevalence of 20.3%, with distinct age-group distributions that are consistent with findings from Turkey, Bangladesh, and Zambia (Ozerol et al., 2005; Chakraborty et al., 2024; Silwamba et al., 2022). The severity of ETEC diarrhea was greater in adults, with more cases of dehydration and a higher need for intravenous (IV) rehydration therapy, emphasizing the significant clinical burden of ETEC in the region.

The seasonal dynamics of ETEC infections observed in this study further support the established understanding that ETEC prevalence exhibits a seasonal peak. Similar to patterns documented in Bangladesh and Egypt (Hosangadi et al., 2019; Mansour et al., 2014), our data indicate that ETEC incidence peaks in the pre-monsoon season, reaching its highest incidence in June before declining during the rainy months. In addition, variations in toxin phenotype expression were observed, with ST-producing ETEC being more prevalent in children and LT-producing ETEC being more prevalent in adults. This finding is clinically significant, as LT-ETEC infections are associated with more severe dehydration and increased hospitalization rates, resembling cholera-like diarrheal illness in North India (Taneza et al., 2004).

Another critical finding of this study is the high prevalence of multidrug-resistant (MDR) ETEC, which raises substantial public health concerns. The study reports that ETEC isolates showed poor response to multiple commonly used antibiotics, including ampicillin, norfloxacin, ofloxacin, doxycycline, furazolidone, tetracycline, azithromycin, and ciprofloxacin. In contrast, they remained susceptible to neomycin, streptomycin, chloramphenicol, amikacin, and gentamicin. The observed 95% treatment failure rate with several first-line drugs underscores the growing challenge of antimicrobial resistance (AMR) and highlights the need for ongoing monitoring. Comparisons with previous studies indicate that MDR ETEC is a rising global issue, with therapeutic efficacy patterns shifting over time (Johura et al., 2024; Huchin et al., 2018). Notably, the reduced effectiveness of fluoroquinolones—commonly reported in India, Bangladesh, and Mexico—is particularly alarming in a tourism-intensive region such as Puri, where travelers may face elevated risk (Huchin et al., 2018; Chakraborty et al., 2001). In addition to ETEC, previous reports from India and neighboring countries have also documented drug resistance trends in other enteric pathogens, such as V. cholerae, Shigella, and Salmonella. The study emphasizes the urgent need to revise local diarrhea treatment protocols in light of the diminished efficacy of fluoroquinolones and macrolides.

The unexpected decline in V. cholerae isolation in 2022, coupled with the increased dominance of ETEC, raises important epidemiological questions. Several hypotheses may explain this shift: (a) Improved sanitation and safe drinking water initiatives may have reduced V. cholerae transmission while still allowing ETEC to spread via direct contact or contaminated food; (b) climatic factors, such as increasing temperatures, may have influenced bacterial survival and proliferation, favoring ETEC over V. cholerae; (c) high historical cholera exposure may have led to increased immunity in the local population, potentially reducing V. cholerae incidence while allowing other enteric pathogens to emerge; (d) microbial competition within the gut may be favoring ETEC under current ecological conditions; and (e) oral cholera vaccine (OCV) campaigns may have provided indirect protection against V. cholerae, inadvertently enabling ETEC to become the predominant pathogen (Samal et al., 2008; Hosangadi et al., 2019).