High Microbial Load and Antibiotic-Resistant Pathogens in Street-Vended Sugarcane Juice: A Public Health Perspective

1. INTRODUCTION

Fruits are an essential component of a healthy diet due to their rich content of vitamins, minerals, dietary fiber, carbohydrates, and phytonutrients. These bioactive compounds have been associated with numerous health benefits, particularly enhanced immune function and protection against various diseases. One of the most common ways to consume as freshly prepared juice [1]. Freshly prepared fruit juices are often act as "rapid energy boosters" because of rapidly absorbed into the bloodstream, providing an immediate energy [2]. The demand for street-vended fruit juices has increased considerably among urban populations due to their affordability, unique taste, variety, and convenience [3]. To maximize customer access, most vendors set-up their stalls in high-traffic public areas. However, the absence of basic sanitary facilities, including access to clean running water, waste disposal systems, and hygienic toilet facilities, raises serious concerns regarding the hygienic conditions of food preparation areas and the personal hygiene practices of vendors [3, 4].

Furthermore, the increasing prevalence of multidrug-resistant food-borne bacteria has exacerbated this public health challenge, contributing to outbreaks linked to contaminated fruit juice consumption [5]. Sugarcane (Saccharum officinarum L.) is a biennial herbaceous plant belonging to the family Poaceae and is primarily cultivated for its sucrose-rich stalks. In Bangladesh, sugarcane is grown on approximately 0.18 million hectares of land, with an average annual production of about 7.3 million tons. Of the total sugarcane produced, approximately 32.36% is used for sugar production, 52.69% for molasses production, and 14.39% for seed and juice production [6]. Sugarcane juice is a nutritious and refreshing natural beverage containing essential vitamins, minerals, simple carbohydrates are readily digestible by the human body [7]. Its popularity is largely attributed to its pleasant taste and excellent thirst-quenching properties. 

Despite its nutritional value, sugarcane juice is highly perishable because of its favorable physicochemical characteristics for microbial growth, including high moisture content, elevated sugar concentration, high water activity, and a near-neutral pH [8]. In Bangladesh, a considerable number of vendors are involved in the extraction and sale of fresh sugarcane juice. However poor sanitation of the sugarcane stalks, extraction equipment, water used during processing, and food-contact surfaces can lead to microbial contamination [9]. Street-vended fruit juices have been found to harbor a variety of bacterial pathogens, including E. coli, Pseudomonas aeruginosa, Salmonella spp., Proteus spp., Klebsiella spp., Enterobacter spp., and S. aureus [10, 11]. Similarly, grape, sweet lime, pineapple, and sapota juices have been reported to contain pathogenic microorganisms such as E. coli, Salmonella spp., Shigella spp., and S. aureus. Moreover, street-vended fruit juices have tested positive for Staphylococcus spp. and fecal coliform bacteria [12].

Consumption of contaminated sugarcane juice poses a significant public health risk by increasing the incidence of foodborne diseases, particularly among nutritionally vulnerable populations. Common bacterial contaminants such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter spp. and Staphylococcus aureus have been associated with Food poisoning, urinary tract infections, Lower respiratory tract infections, gallbladder infections, middle ear infections, and healthcare-associated bacteremia [13]. In addition, E. coli is a major etiological agent of diarrhea, pyogenic infections, septicemia, and other potentially life-threatening conditions [14]. The detection of Pseudomonas spp., Staphylococcus spp. and Escherichia coli is particularly alarming because these microorganisms can produce enterotoxins that may cause foodborne illnesses in freshly prepared sugarcane juice is a matter of concern [15, 16].

Despite the popularity of sugarcane juice and the documented hygiene deficits of street-vending practices, data specifically from southern Bangladesh remain scarce. Gopalganj, a growing urban center, hosts numerous juice vendors whose products are consumed daily by students, workers, and the general public. Systematic assessment of bacterial load, species composition, and resistance profiles in this locality is therefore warranted.

Therefore, the present study aimed to- quantify total viable bacteria in street-vended sugarcane juice collected from four sites in university areas; isolate and phenotypically identify the bacterial genera and determine the antimicrobial susceptibility patterns of the recovered isolates. The findings are expected to inform local food-safety interventions. Also, can contribute to the broader understanding of street-food-associated antimicrobial resistance and risk of foodborne disease outbreaks. 

2. MATERIALS AND METHODS

2.1. Sample collection

The study was conducted during April-May 2024 in the Gopalganj city area in Bangladesh. A total of four street-vended sugarcane juice samples were collected from different vendors. Four freshly extracted sugarcane juice samples (approximately 20 mL each) were aseptically collected from different street vendors at four locations in the city; GSTU Campus (BM 1), Nobinbag (BM 2), Bangabandhu College More (BM 3), and Chourangi (BM 4). Samples were transferred into sterile Falcon tubes, immediately placed in an icebox, transported to the laboratory, and stored at 4°C. All microbiological analyses were completed within 24 h of collection.

2.2. Sample preparation

Each sugarcane juice sample was serially diluted using sterile phosphate-buffered saline (PBS). Briefly, 1 mL of each sample was aseptically transferred into 9 mL of sterile PBS to obtain a 10⁻¹ dilution. Subsequent serial tenfold dilutions were prepared as required for microbiological analysis.

2.3. Total viable count (TVC)

The total viable bacterial count was determined using the spread plate technique. Briefly, 50 µL aliquots from appropriate serial dilutions were spread evenly onto Plate Count Agar (PCA) plates using a sterile spreader. The inoculated plates were incubated at 37°C for 24 hours. After incubation, plates containing 30–300 colonies were selected for enumeration, and the bacterial load was expressed as colony-forming units per milliliter (CFU/mL) of the sample.

2.4. Isolation, purification, and phenotypic identification

Bacteriological analysis was performed following standard microbiological procedures for the isolation of aerobic bacteria. Each sugarcane juice sample was first enriched in Nutrient Broth and incubated overnight at 37°C. The enriched cultures were subsequently streaked onto Nutrient Agar plates using the streak plate technique to obtain isolated colonies. Pure cultures were obtained through repeated sub-culturing until morphologically homogeneous colonies were observed. The purified isolates were then inoculated onto selective and differential media, including Mannitol Salt Agar (MSA), MacConkey Agar, Eosin Methylene Blue (EMB) Agar, and Salmonella–Shigella (SS) Agar, and incubated at 37°C for 24 hours to facilitate bacterial identification. The isolated bacteria were identified according to the standard procedures recommended by the International Commission on Microbiological Specifications for Foods (ICMSF). Identification was based on colony morphology, Gram staining, and a series of biochemical tests included motility, indole production, oxidase, citrate utilization, coagulase, and catalase tests. The overall laboratory procedure illustrated in Figure 1. 

Figure 1.  Laboratory workflow for microbiological analysis of street- vended sugarcane juice samples.

2.5. Antibiogram study

The antimicrobial susceptibility of the isolated bacterial strains was determined using the Kirby–Bauer disc diffusion method on Mueller–Hinton agar (Difco) following the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Commercially available antibiotic discs were placed on the inoculated agar surface, and the plates were incubated at 37°C for 18–24 hours. After incubation, the diameters of the inhibition zones around each antibiotic disc were measured in millimeters. The bacterial isolates were classified as susceptible, intermediate, or resistant according to the CLSI interpretative criteria.

2.6. Quality control 

All media and reagents were prepared according to manufacturers’ instructions and sterility-checked before use. Positive and negative controls were included in biochemical and susceptibility testing where appropriate. Results were recorded and tabulated for descriptive analysis; no inferential statistics were applied owing to the limited sample size.

3. RESULTS

3.1. Total viable count (TVC) results for the microbial load 

Table 1 presents TVC of street-vended sugarcane juice samples collected from different vendors. Among the samples analyzed, the sample (BM 1) collected from the Gopalganj Science and Technology University (GSTU) campus exhibited the highest bacterial load, with a TVC of 4.2 × 106 CFU/mL followed by sample BM4 (3.8 × 106). 

Table 1. Microbial load by total viable count. 

Note. BM 1 = GSTU Campus; BM 2 = Nobinbag; BM 3 = Bangabandhu College More; BM 4 = Chourangi.

3.2. Isolation and cultural characteristics

Figure 2 illustrating the morphological examination on selective media identified distinct bacterial growth patterns across the 12 purified isolates.

Figure 2. Bacterial growth patterns observed on street-vended sugarcane juice samples collected from different vendors after cultivation using the spread-plate technique. Here, (A) Citrobacter spp., (B) Escherichia coli (C) Staphylococcus spp. (D) Klebsiella spp. (E) Enterobacter spp.

A total of 12 bacterial isolates belonging to six presumptive groups were recovered from four samples of street-vended sugarcane juice (Table 2). Staphylococcus spp. and Klebsiella spp. were the most frequently detected organisms, each present in 75% (3/4) of the samples (3 isolates each). Escherichia coli and Enterobacter spp. were isolated from 50% (2/4) of the samples (2 isolates each), while Citrobacter spp. and Salmonella spp. were each recovered from a single sample (25%; 1 isolate each). Colony morphologies on the respective selective/differential media were consistent with the presumptive identifications of these genera.

Table 2. Results of isolation of bacteria from street-vended sugarcane juice.

Note: EMB= Eosin Methylene Blue; SS= Salmonella–Shigella. (Representative agar plate photographs for culture characterization were not captured during primary laboratory testing).

3.3 Biochemical characterizations

Biochemical characterization of the 12 isolates (Table 3) confirmed the presumptive identifications obtained from selective media. Three isolates were identified as Staphylococcus spp. (Gram-positive, catalase-positive, coagulase-positive), three as Klebsiella spp. (Gram-negative, VP-positive, urease-positive, non-motile), two as Enterobacter spp., two as Escherichia coli (indole-positive, MR-positive), and one each as Citrobacter spp. and Salmonella spp. (H₂S-positive on TSI). All Gram-negative isolates were oxidase-negative and catalase-positive, with TSI reactions consistent with their respective genera.

Table 3. Biochemical test results. 

Note: MR= Methyl Red; VP= Voges–Proskauer; TSI=Triple Sugar Iron (Y = Yellow/Acidic, R = Red/Alkaline); N/A: Not Applicable

In Figure 3, E. coli, Salmonella spp., and Staphylococcus spp. tested positive in the methyl red (MR) test, whereas Klebsiella spp. tested negative. All isolates were positive for the catalase test, as evidenced by gas bubble formation. Except for Staphylococcus spp., all isolates tested negative in the oxidase test, showing no color change. The Voges-Proskauer (VP) test yielded positive results for Salmonella spp. and Klebsiella spp., whereas E. coli and Staphylococcus spp. showed negative results. 

Figure 3: Biochemical test results: A= MR control, B=MR positive=MR negative, D=VP control, E= VP positive, F= VP negative, G= citrate control, H= Citrate positive, I= Citrate negative, J= TSI control K, L = TSI yellow slant and butt and gas positive, M=Motility and Indole control, N = Motility and Indole positive, O= Motility and Indole negative, P= Urease control, Q= Urease positive, R= Urease negative, S, T= Coagulase positive, U= Catalase positive, V= Catalase negative.

3.4. Antimicrobial susceptibility profiles

Figure 4 shows representative antibiotic susceptibility testing results on Mueller-Hinton agar using the disc diffusion method. Clear zones of inhibition surrounding antibiotic discs indicate susceptibility of the tested isolates. (A) Enterobacter spp.; (B) Staphylococcus spp.; (C) Klebsiella spp. 

Figure 4. Representative images of antibiotic sensitivity test. Different antibiotic discs were placed on Muller-Hinton agar media and cultured for bacterial growth. A= Enterobacter spp., B= Staphylococcus spp., C= Klebsiella spp.

The average results of the antibiotic susceptibility assay are presented in (Table 4). Co-trimoxazole and Gentamicin were found to be effective against all six bacterial isolates. Enterobacter spp. and Salmonella spp. were resistant to Doxycycline and Tetracycline. Ciprofloxacin was resistant Staphylococcus spp. and intermediate for Enterobacter spp. Furthermore, all isolates exhibited resistance to Amoxicillin, Erythromycin, Ampicillin, and Metronidazole.   

Table 4. Antimicrobial susceptibility profiles of bacterial isolates from sugarcane juice samples.

Note: Values represent the calculated zones of inhibition (mm). I = intermediate, S = sensitive, and R = resistant, indicating the responses of the bacterial isolates to the respective antibiotics [17].

4. DISCUSSION

The contamination profile observed street-vended sugarcane juice in Gopalganj contaminated by various types of bacteria. The more alarming finding is the presence of fecal coliforms in all samples, as they are known to only survive briefly in the environment outside the gut, and their presence suggests a contamination source that is there more recently, or at least present at the time of sampling, as opposed to historical or incidental contamination [18,19]. The "elevated" microbial counts (3.2×10⁶–4.2×10⁶ CFU/mL) are not just "higher" than normal relative to any particular pathogen, directly associated with symptomatic foodborne illness as a class, stand-alone from each pathogen's virulence [20]. Added to this, the identified genera like Klebsiella, E. coli and Salmonella are well recognized causes of gastroenteritis, urinary tract infection and invasive bloodstream infection, respectively, and S. aureus includes a pathogen that can cause intoxication without being viable at the time of ingestion. Other studies also have been documented in street foods sold near educational institutions and in retail meat samples from local markets in Bangladesh, underscoring inadequate hygienic practices and antimicrobial resistance circulating in the informal food sector [21-23].

Beyond the elevated microbial load, the antimicrobial-resistance patterns observed are of particular concern. This pattern of high resistance to ampicillin, erythromycin, amoxicillin and metronidazole and high susceptibility to gentamicin, co-trimoxazole and ciprofloxacin is alarming for public health. This resistance pattern is likely attributable to the widespread availability of these antibiotics at low cost, their frequent sale without prescription, and their common use as first-line agents in both human and veterinary healthcare sectors in Bangladesh. In this context, the resistance profile of street-vended juice isolates is an indirect marker of the local antimicrobial consumption profile, already reported in many countries like India, Nigeria, and Ethiopia [13, 24, 25]. 

A question, the results do not answer, is whether sugarcane juice is a passive reservoir of resistance that is already present in the environment or an active source for resistance gene exchange? The characteristics of sugarcane juice high nutrient content, warm temperature, frequent human handling offer a potential scenario for horizontal transfer between co-occurring genera, which would allow the transfer of resistance determinants regardless of the resistance status of any individual plant's species [26, 27]. Then contaminated juice is not only a reflection of the antimicrobial resistance issue, it also can be an amplifier as it introduces resistant microbes and mobile resistance elements into a broader human population via an unregulated, high turnover food product [28]. The present study cannot confirm whether gene transfer is occurring; future work using plasmid profiling or whole-genome sequencing would be needed to answer this question.

The findings must be interpreted in light of several limitations. The sample size was small (n = 4), restricting statistical power and generalizability. Bacterial identification relied solely on cultural and biochemical criteria and therefore remains presumptive; molecular confirmation was not performed. Minimum inhibitory concentration (MIC) assays were not performed, and the observed inhibition-zone diameters were not correlated with specific antimicrobial resistance genes. Consequently, the study could not determine the underlying molecular mechanisms responsible for the observed resistance phenotypes. Despite these constraints, the consistent recovery of high bacterial loads and resistant isolates across all sampling sites provides clear evidence of inadequate hygienic practices and a tangible public-health risk. Strengthening vendor education on basic food-hygiene principles, ensuring access to potable water, enforcing equipment sanitation, and implementing periodic microbiological surveillance are essential measures to reduce consumer exposure

5. CONCLUSIONS

This study demonstrates that street-vended sugarcane juice sold in Gopalganj, Bangladesh, is frequently contaminated with potentially pathogenic bacteria, with microbial loads exceeding the permissible limits established for fruit juices in many countries. The bacterial isolates also exhibited diverse antibiotic resistance patterns, including resistance to commonly used antimicrobial agents, raising concerns about the potential dissemination of antimicrobial-resistant bacteria through ready-to-consume beverages. These findings suggest that the juice is often prepared, handled, and marketed under inadequate hygienic and sanitary conditions, likely due to the use of contaminated water, poor equipment sanitation, and insufficient knowledge of food safety practices among vendors. From a public health perspective, the consumption of contaminated sugarcane juice may increase the risk of foodborne illness and contribute to the spread of antimicrobial resistance within the community. Strengthening food safety regulations, improving vendor hygiene training, ensuring access to safe water, and implementing regular microbiological surveillance are essential to minimize these risks and protect consumer health.

ACKNOWLEDGEMENT

The authors would like to thank all co-authors for their valuable contributions to the conceptualization, drafting, and critical revision of this manuscript. We also thank the University administration for its institutional support of this study. The authors further acknowledge that artificial intelligence (AI) tools were used solely for text editing and reference formatting, in accordance with the journal's editorial policies. 

FUNDING STATEMENT

The authors declare that they received no financial support or funding for the research, authorship, and/or publication of this article.

CONFLICT OF INTEREST

The authors declare that they have no known competing financial or personal interests that could have appeared to influence the work reported in this paper. 

ETHICAL CONSIDERATION

This study did not involve human participants or animals; therefore, ethical approval was not required.

AUTHOR CONTRIBUTIONS

Md. Sarafat Ali: Conceptualization, methodology, formal analysis, writing original draft, review & editing.  B M Tanjim Abid: Investigation, formal analysis, review & editing.  All authors have read and approved the final manuscript.

AVAILABILITY OF DATA AND MATERIAL

The data that support the findings of this study are available in the article. Further details may be obtained from the corresponding author upon request.

DECLARATION OF AI USE

The AI tools were not used to generate scientific content, interpret data, or draw conclusions. All literature selection, data interpretation, and final manuscript preparation were performed entirely by the authors, who accept full responsibility for the accuracy and integrity of the work.

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