Screening of multidrug-resistant coliforms from fruit juice and their significant correlations with the developmental deformities of Artemia salina (Brine shrimp)

Screening of multidrug-resistant coliforms from fruit juice and their significant correlations with the developmental deformities of Artemia salina (Brine shrimp) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Screening of multidrug-resistant coliforms from fruit juice and their significant correlations with the developmental deformities of Artemia salina (Brine shrimp) Md. Nazmul Haque, Afrin Priya Talukder, Shaila Shaimun Diba, Md. Salah Uddin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6737003/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Microbial spoilage of fruit juice is one of the major concerns for human infections in Bangladesh. In this context, local and packaged fruit juices were microbiologically assessed based on lactose-fermenting ability. The isolates were tested to analyze acute toxicity against the developmental stages of Artemia salina and correlations between the bacterial dose duration and brine shrimp’s abnormalities. The morphological, physiological, biochemical, and 16S rRNA sequencing revealed that isolate A was Acinetobacter sp. (lactose-non-fermenting, sample-6) and B was Klebsiella sp. (lactose-fermenting, sample-9). The developmental toxicity of both bacteria induced delayed germination of Artemia cysts and after 48 hours, missing antenulla, abnormal eye, contused mandibles, absent swimming legs, and damaged ovaries were recorded. The Duncan Multiple Range Test confirmed the highest toxicity of Acinetobacter sp. compared to Klebsiella sp. The time of death (tD 50 ) was 5 hours for Acinetobacter sp. and 6 hours for Klebsiella sp. The lethal concentration (LC 50 ) values were 61.07 ± 0.19µl and 69.99 ± 0.21µl, respectively. Pearson correlations revealed defective traits were positively correlated with the increased coliform’s exposure duration to Artemia salina and multidimensional test confirmed complete separation between control and treated Artemia . Both bacteria were multidrug-resistant. This study suggests that awareness should be raised to eradicate microbial health hazards. fruit juice multidrug-resistant (MDR) Artemia salina deformities pearson correlation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Fruits are an essential ingredient of a well-balanced aliment and fruit juices are widely consumed as a source of essential minerals, nutrients, antioxidants, and dietary fibres. Fruit juices are nutrient-dense beverages that taste great and have several health advantages (Suaads and Hamed 2008 ). Juice contains significant quantities of vitamins C and E along with antioxidants, and has a nice flavour and aroma. These essential restorative properties may reduce the risk of many diseases (Abbo et al. 2006 ). Notably, fruit juices play immense roles in human body detoxification, prevention of vitamin A and vitamin C deficiencies as well as enhancement in the blood lipid profile of individuals with hypercholesterolemia. Regular intake of fruit juice is linked with reducing the chances of developing cataracts, Alzheimer's disease, cancer, heart disease, stroke, as well as other anomalies associated with aging (Liu 2003 ). For instance, Mango juice contains components like phytochemicals and antioxidants (Liu et al. 2016 ), which are very helpful for curing serious ailments as well as cancer. A glass of chilled sugarcane juice not only satisfies our thirst but also rejuvenates us. Nevertheless, even if fruit juice has a high nutritional value, the production and processing practices in developing countries may not reach suitable safety levels and pose consumers at risk of illness. Foodborne diseases are one of the major reasons for global fatalities, predominantly in developing countries (Oladipo & Adejumobi 2010 ). Several studies reported food-borne illness caused by the consumption of fruit juices and the prevalence of coliforms on fruit surfaces is a clear indication of fecal pollution which can be detrimental to human health (Reddy et al. 2016 ; Padamadan et al. 2016 ). A wide range of diverse microorganisms known as "coliform bacteria" that are utilized as indicator organisms for assuring the presence of pathogenic contagious microbes, especially bacteria in foods and water. The key characteristics of coliform include its aerobic nature, gram-negative status, lack of spore formation, rod-like morphology, and lactose fermentation (Isfahani et al. 2017 ). Juice processing with unhygienic water can be a most important vehicle for microbial shipment, for instance fecal coliforms, coliforms as well as fecal Streptococci etc. (Tasnim et al. 2010 ). Juices are latent sources of harmful bacteria including Staphylococcus aureus, Salmonella sp., E. coli 0157, Shigella sp., Erwinia sp., Klebsiella pneumonia , Enterobacter sp., Acinetobacter sp., Pseudomonas sp., and Alicyclobacillus sp. (Lewis et al. 2006 ). For instance, Acinetobacter sp. is frequently responsible for illnesses including meningitis, skin and soft tissue infections, bacteremia, respiratory and urinary tract diseases (Oberoi et al. 2009 ). Furthermore, the most prevalent Enterobacteriaceae pathogenic bacteria that cause nosocomial infection outbreaks is Klebsiella pneumoniae , which is especially common in critical care, burn, oncology, and neonatal units (Correa et al. 2013 ). Prior research has revealed that fruit juices from street vendors as well as those in packs are contaminated with harmful microbes (Chen et al. 2010 ). In Bangladesh, street-vented and packed fruit juices are popular among people because they fulfill nutritional requirements at a reasonable price. Nonetheless, the easy availability of fruit juices is a familiar source of pathogenic bacterial transmission in developing countries like Bangladesh (Sarker et al. 2013 ; Das et al. 2012 ). There are several reasons behind this microbial contamination. Firstly, lack of personal hygiene, conventional processes of juice preparation, and inappropriate preserving temperature are the most important factors (Rane 2011 ). Additional justifications include fruit flies, filth, and improperly cleaned tools like cutters and knives, as well as direct contamination from rotting fruits (Reddy et al. 2016 ; Khan et al. 2015 ). Because of improper handling and serving procedures, street-vended fruit juices are frequently linked to diarrheal illnesses in Bangladesh (Bello et al. 2013 ). Due to these negative consequences of consuming contaminated fruit juices, it is indispensable to measure the coliform’s acute toxicity in a model organism to address the microbial health hazards risk from fruit juice. Artemia salina (brine shrimp) is a widely recognized crustacean found in seawater environments (saline lakes and seas) and a vital food source for fish and other aquatic invertebrates (Zhu et al. 2018 ). The model organism has been widely used to identify the toxic effect of bacteria, chemicals due to its year-round commercially availability of dried cysts, low cost, short life cycle and easy to maintain in lab at in vivo conditions (Faimali et al. 2012 ). This assay is regarded as a reliable way to assess the level of toxicity in plant extract (Solis et al. 1993 ), and evaluation of toxic cyanobacteria (Lahti et al. 1995 ) and heavy metals (Ates et al. 2017 ). Previous studies revealed a high association between the cytotoxicity directed against certain tumor cell lines and the toxic activities in the brine shrimp experiment (Anderson et al. 1991 ) as well as the hepatotoxic activity (Kiviranta et al. 1991 ). Thus, Artemia salina experiment is a more feasible way compared to cell lines assays for in vivo acute toxicity evaluation (Piccardi et al. 2000 ). Therefore, our research aimed to investigate coliform bacterial availability in collected fruit juice (locally and commercially) and coliform’s acute toxicity detection through Artemia Salina . The aim also includes a statistical justification of significant correlation between coliform and coliform-treated abnormalities of Artemia (during stationary and exponential phase) and their antibiotic-resistant status to understand the possible public health concern. 2. Materials and methods 2.1. Sample Collection Twelve samples of fruit juice were taken from different areas of the Rajshahi metropolitan area (site-1: Laxmipur, site-2: Railway station, site-3: University campus, and site-4: Shaheb bazar) in May 2022. Among the twelve samples, Sample 1 to Sample 7 were street-vended fruit juice samples, namely papaya juice for Sample 1, orange juice for Sample 2, lemon juice for Sample 3, bael juice for Sample 4, watermelon juice for Sample 5, sugarcane juice for Sample 6, and pineapple juice for Sample 7. The remaining five samples were packed mango juices samples identified as Sample 8 to Sample 12). Subsequently, all of the samples were taken directly to the Microbiology Laboratory of the Department of Genetic Engineering and Biotechnology at the University of Rajshahi to evaluate their microbiological quality. 2.2. Isolation of Coliform Bacteria and Optimization of Their Growth Parameters The samples of fruit juices were collected as a source of inoculum, and droplets of juice were used for plating on MacConkey agar medium, which contains the following substances: (Lactose monohydrate 10.0 g/L, peptones 3.0 g/L, bile salts 1.5 g/L, sodium chloride 5.0 g/L, crystal violet 0.001 g/L, and Agar 13.5 g/L at pH = 7.0) separately. The plates were subsequently incubated at 37°C for a duration of 24 hours. Two coliform bacteria were selected and marked as A and B based on lactose fermentation capacity and healthy colony morphology. These two bacteria showed highest CFU (colony forming unit) among all other bacteria which were 8.5×10 9 cfu/ml and 6.8×10 7 cfu/ml for Isolate A and Isolate B, respectively. Lastly, strains that had been isolated as pure cultures were kept for use in future research. Different temperatures and pH influence bacterial growth. In this experiment, the pH was varied at 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0, while the temperature was 25, 30, 35, and 40°C. In both cases, the optical density of the bacterial liquid cultures was recorded with a double-beam spectrophotometer (Analytk Jena AG, Germany), at 660 nm. 2.3. Morphological and Biochemical Characterization of Isolated Bacteria Numerous morphological techniques, such as Gram staining and motility, as well as biochemical assays, including methyl red, catalase, urea hydrolysis, Simmons citrate, and TSI test, were used to characterize the isolates. To differentiate between gram-negative and gram-positive bacteria by analysing their cell wall structures, gram staining (0.5% Crystal violet solution, 1% Lugol's iodine solution, and 1% safranin solution) was conducted. Motility test (Agar 4.0 g/L, NaCl 5.0 g/L, and Peptone 10.0 g/L) was used to observe bacterial planktonic status. Among the biochemical test, catalase test (0.5 g/L of yeast extract, 1 g/L of peptone, and 2.4 g/L of agar) was performed to check hydrogen peroxide detoxification ability of aerobic bacteria. Methyl red test (Basal salt media: 2.34g of K 2 Cr 2 O 7 , 1.33g of KH 2 PO 4 , 0.02g of MgSO 4 . 7H 2 O, 1.00g of (NH 4 ) 2 SO 4 , 0.50g of NaCl, 0.10g of yeast extract, and 1.00g of glucose) was used to know the ability of microorganism’s mixed acid fermentation. Along with these tests, the citrate test (1.05 g/L of bromothymol blue, 1.0 g/L of ammonium dihydrogen phosphate, 1.0 g/L of dipotassium phosphate, 2.0 g/L of sodium citrate dehydrate, 5.0 g/L of sodium chloride, 15.0 g/L of agar, and 0.20 g/L of magnesium sulphate heptahydrate) was done for citrate utilizing ability, urease test (Agar 15.0 g/L, 0.012 g/L of phenol red, 1.0 g/L of dextrose, 1.20 g/L of disodium phosphate, 0.80 g/L of monopotassium phosphate, 5.0 g/L of sodium chloride, and 1.0 g/L of peptic digest of animal tissue) for urease enzymatic activity, and the triple sugar iron agar test was used to determine whether lactose, sucrose, and dextrose could ferment. The morphological and biochemical tests were conducted as per standard protocols (Collin et al.1995). 2.4. Molecular Identification of Isolated Bacteria The following procedures were used to identify the isolated bacteria at the molecular level: Extracting the chromosomal DNA, amplifying the 16S rRNA gene, purifying the PCR product, cycle sequencing with purifying the cycle sequencing product, detecting the nucleotides, and sequence analysis. The CTAB technique was used to extract the bacterial genomic DNA described by Edwards et al. (Edwards et al. 1991 ). After extraction, The NanoDrop Spectrophotometer (Thermo Scientific, USA; ND2000 series) was used to quantify DNA. PCR was used to amplify the 16S rRNA gene following the instructions provided by Loffler et al. (Loffler et al. 2000) using 16s F and 16s R (Sigma, USA) universal primers. The primer sequences are provided in the supplementary file (Table S1 ). All PCR reagents were used from the Promega (Catalogue no. M7432, Promega, USA). The composition of the PCR Reaction mix (Total volume is 25 µl) are in supplementary (Table S2). The reagents were mixed gently, and short spin was done. PCR amplification was done by Gene Atlas (G2; Astec, Japan). Cycling parameters for PCR analysis in supplementary. After PCR amplification, samples were run in agarose gel (Catalogue no. V3125; Promega, USA) for visualization of the band. In this process, Promega, USA's 1 kb DNA ladder (Catalogue no. G5711), TAE buffer (Catalogue no. V4251), and ethidium bromide solution (Catalogue no. H5041) were utilized. In order to purify the PCR product, 40 µl of the product was combined with 160 µl of sterile water, resulting in a final amount of 200 µl. Following that, the 200 µl was transferred to the YM-100 rapid spin columns and centrifuged for 10 minutes at 500 xg. The columns were moved into fresh tubes for the DNA elution process, and added 25 µl of sterile water. Next, for five minutes, the tubes were centrifuged at 1000 xg. Genetic Analyzer (3500, Thermo Scientific, USA) was used for the sequencing of the PCR-purified product. After that, the bioinformatics tool Chromas was used to edit the sequences. Then the BLASTN algorithm ( http://www.ncbi.nih.gov ) was used to determine the homology between the 16S rRNA gene sequence and the gene sequences of other organisms. 2.5. Evaluating the Toxic Effects of Isolated Bacteria on the Developmental Stages of Artemia salina An acute toxicity assay was conducted to compare the cellular toxicity of coliform bacterial strains obtained from fruit juice at two developmental stages of Artemia salina : the stationary and the exponential phases. 2.5.1. Stationary Phase Assays for intoxication were conducted using 6-well plates, with 10 ml of filtered seawater in each well. Ten Artemia salina cysts were co-cultured and incubated with isolated bacteria that had grown overnight at an optical density of 0.5, with three replications at an abundance of 200 cells ml − 1 . A control containing brine shrimp without isolated bacteria was also implemented in three times. In this case, a mineral salt (MS) liquid medium was used for bacterial culture. Every six hours during the 48-hour incubation period, the germination of the cysts was inspected, and any significant anomalies were noted using an inverted microscope (USA: LABOMED CXL). 2.5.2. Exponential Phase The experiment was performed using 6-well plates, with each well containing 10 ml of filtered seawater, and this phase was observed for up to 7 hours. The experiment was carried out in triplicate with a control group and involved incubating six individuals of Artemia salina for seven hours with overnight cultured isolated bacteria (O.D. at 0.5) at an abundance of 200 cells ml − 1 . Every hour after incubation, the survival of individuals and significant anomalies were examined under an inverted microscope (LABOMED CXL, USA). 2.6. Determination of tD 50 In the case of the exponential phase, the tD 50 (the time when 50% of Artemia salina died) value was calculated following the protocol outlined by Neves et al. (Neves et al.2017) [30] 2.7. Determination of LC 50 The lethality bioassay of brine shrimp is considered a helpful method for conducting an initial toxicity assessment (Solis et al. 1993 ). Nauplii were obtained by hatching Artemia salina eggs in simulated seawater. To evaluate the cytotoxic activity of the isolated bacteria on brine shrimp, six autoclaved test tubes were prepared, and 10 ml of artificial seawater containing 10 nauplii were introduced into each tube. Finally, 25, 50, 75, 100, 125, and 150 µl of the previously cultured bacterial samples were sequentially added to the test tubes using a micropipette. The test tubes exposed to light and incubated at ambient temperature for 24 hours to monitor the cytotoxic effects. After the 24-hour period, the number of survivors was recorded. This data was then analyzed using a basic probit analysis tool to calculate the LC 50 value with 95% confidence intervals. 2.8. Statistical analysis for significant changes and correlation between coliform doses and Artemia abnormality For statistical analysis, the Duncan Multiple Range Test (DMRT) was conducted using Statistical Analysis Software (SAS, version 9.1.3) to compare the means of three replicates between groups. To visualize the significant changes through MDS, the data of Artemia cysts treated with coliform bacteria Isolate A and isolate B were normalized and analyzed separately for the stationary and exponential phases. ANOSIM (Analysis of Similarity) was performed to determine significant differences in abnormalities between coliform-treated Artemia and untreated Artemia using Primer E (Version 7). To measure the Pearson correlations between the abnormal features of treated Artemia and the duration of dose, heatmaps were generated using the heatmap package in R (version 4.1.3). 2.9. Antibiotic Sensitivity Test The antibiotic sensitivity profile of isolated coliform bacteria was assessed using the Kirby-Bauer disc diffusion technique (Bauer et al. 1966 ). In this study, both Luria broth (LB) liquid and LB agar were utilized as culture media to facilitate bacterial growth. Commercially available and commonly recommended antibiotics such as penicillin (10 µg per disc), amoxicillin (10 µg per disc), erythromycin (15 µg per disc), ampicillin (10 µg per disc), kanamycin (30 µg per disc), tetracycline (30 µg per disc), ceftazidime (30 µg per disc), gentamycin (10 µg per disc), ciprofloxacin (5 µg per disc), cefuroxime (30 µg per disc), cefixime (5 µg per disc), Doxycycline (10 µg per disc) were used as antibiotic discs, placed in the center of the corresponding plates, and incubated overnight at 37°C. Following an overnight incubation period, the zones were examined and measured using the mm scale. 3. Results 3.1. Isolation and Optimization of Growth Characteristics of Isolated Bacteria Coliform bacterial strains were isolated from the collected fruit juices through screening on MacConkey agar media and the highest number of colonies were found in the following pattern: Sample 6 > Sample 9 > Sample 4 > Sample 2 > Sample 1 > Sample 12 > Sample 7 > Sample 5 > Sample 10 > Sample 3 (Fig. 1 ). Out of the twelve samples, Sample 6 (street juice with a white colony) had the highest number of coliform colonies, and Sample 9 (packed juice screened by a pink colony) was chosen for additional tests. However, Samples 8 and 11, both packed juices, showed no growth on MacConkey agar media (Fig. 1 ). According to our findings, the maximum growth of bacteria was observed in isolates A (Figure S1 ) and B (Figure S2) at pH 7.0 and 7.2, respectively. The optimal temperature for bacterial growth was 35°C for isolate A (Figure S3) and 37°C for isolate B (Figure S4). 3.2. Molecular Profiling of Isolated Bacteria Following 16S rRNA gene sequencing and editing, the sequences were compared to those of other organisms that had previously been uploaded to the GenBank database Some sequences with notable identities were found in the Gen Bank, and isolate A showed a significant 97% similarity with Acinetobacter sp. (Accession no. LC437022), while isolate B showed a 98% similarity with Klebsiella sp. (Accession no. LC552682). The PCR band of the chromosomal DNA and the DNA quantification analysis are shown in Figs. S5-S7, respectively. 3.3. Evaluating the Toxicity of Isolated Bacteria on the Developmental Stages of Artemia salina 3.3.1. Stationary Phase After 16 hours, Artemia cysts treated with both isolates ( Acinetobacter sp. and Klebsiella sp.) showed no germination (Fig. 2 ). Next to 24 hours, both bacteria-treated cysts showed delayed germination (10% germination rate) compared to control (above 65% germination rate). Following the recorded intervals at 36hr, and 48hr, the germination rate of treated groups was lower than 30%, and 40% respectively, while the control group showed more than 80% germination rate in both intervals. Major abnormalities were recorded separately including missing antennules, abnormal eyes, absent swimming legs, and a damaged ovary under a LABOMED CXL microscope (USA), as depicted in Fig. 3 . 3.3.2. Exponential Phase The toxic effects of two coliform isolates were examined on Artemia salina after germination. Treatments were applied in triplicate for each bacterium, along with controlled maintenance. All of the isolates exhibited the highest level of survival after an hour, and no significant variation was observed between the treatments and the control group (Figure. 4). Acinetobacter sp. exhibited lower survival after 2 hours compared to Klebsiella sp., although the difference between the two isolates was not statistically significant when compared to the control (Fig. 4 ). We observed that, between 3 to 7 hours, the survival rate of Artemia decreased over time, resulting in a variation that differed significantly from the control (Fig. 4 ). After 8 hours, no Artemia survived in the presence of Acinetobacter sp. , and the variation was statistically significant (Fig. 4 ). Significant variations were also noticed using a LABOMED CXL microscope (USA), as illustrated in Fig. 5 . 3.4. Determination of tD 50 The tD 50 value for both Acinetobacter sp. and Klebsiella sp. was recorded during the exponential phase, which lasted 5 hours for Acinetobacter sp. and 6 hours for Klebsiella sp. 3.5. Determination of LC 50 The cytotoxic effect of isolated bacteria was examined at different concentrations of 25, 50, 75, 100, 125, and 150 µl in this current investigation. The LC 50 value for Acinetobacter sp., determined through the brine shrimp lethality bioassay, was 49.42 ± 0.21 µl, with the regression equation Y = 2.028x ± 1.565. The 95% confidence interval was between 31.11 and 78.49 µl after 24 hours of exposure (Table 1 , Figure. S8). In contrast, the value for Klebsiella sp. was 69.99 ± 0.21 µl/ml, with a regression equation of Y = 2.390x ± 0.589. The 95% confidence limits ranged from 50.11 to 97.79 µl/ml after 24 hours of exposure (Table 1 and Figure S10). Additionally, the mortality rates of Artemia salina induced by the isolated bacteria are presented in Figures S9 and S11, respectively. Table 1. After a 24-hour exposure, the LC50 values, 95% confidence limits, regression equations, and Chi-square values for the isolated bacteria tested against brine shrimp nauplii are as follows: Test sample LC 50 (μl) 95% Confidence limits (μl) Regression equation χ 2 value (Degrees of freedom) Acinetobacter sp. 61.07±0.19 43.73 to 85.29 Y =2.532x±0.477 0.585 (4) Klebsiella sp. 69.99±0.21 50.11 to 97.79 Y =2.390x±0.589 3.6. Pearson Correlation Between Dosses and Abnormality of Artemia salina Using Pearson correlation, anomalies of treated Artemia after 48 hours were found to be negatively correlated with controls in the case-stationary phase (Fig. 6 a). Similar significant correlations were also detected during the exponential phase (Fig. 6 b). Moreover, the ANOSIM and ordination MDS identified the significant changes and separation between controls and treated Artemia’s abnormalities (Fig. 7 a and 7 b; Table S3 for ANOSIM). 3.7. Antibiotic Sensitivity Test The sensitivity and resistance patterns of the isolated Acinetobacter sp. confirmed that the isolated bacterium was resistant to ciprofloxacin, doxycycline, tetracycline, cefixime, ceftazidime, and penicillin (Table 2 ) but susceptible to cefuroxime, kanamycin, gentamycin, amoxicillin, and erythromycin. On the contrary, Klebsiella sp. was resistant to penicillin, tetracycline, doxycycline, and cefixime while susceptible to amoxicillin, kanamycin, ceftazidime, gentamycin, ciprofloxacin and cefuroxime (Table 2 ). Table 2. Antibiotic resistance profile of isolated bacteria. Name of Antibiotic Zone of inhibition (mm) Resistant pattern Acinetobacter sp. Klebsiella sp. Acinetobacter sp. Klebsiella sp. Penicillin 8 mm 9 mm Resistant Resistant Amoxicillin 19 mm 17 mm Susceptible Susceptible Erythromycin 22 mm 14 mm Susceptible Intermediate resistant Ampicillin 11 mm 15 mm Intermediate resistant Intermediate resistant Kanamycin 16 mm 20 mm Susceptible Susceptible Ceftazidime 10 mm 18 mm Resistant Susceptible Gentamycin 17 mm 22 mm Susceptible Susceptible Tetracycline 9 mm 10 mm Resistant Resistant Doxycycline 7 mm 8 mm Resistant Resistant Ciprofloxacin 8 mm 23 mm Resistant Susceptible Cefuroxime 16 mm 17 mm Susceptible Susceptible Cefixime 6 mm 9 mm Resistant Resistant Note: Resistant=15 mm 4. Discussion Fruit juices are a popular source of nutrients among people in developing countries like Bangladesh. The reason behind this is that fruit juices are delicious, accessible at low prices, and convenient for the consumers (Ohiokpehai 2003 ). Notably, the detection of coliform in fruit juice is not only permissible according to safe food consumption standards (Andres et al. 2004) [33], and regulations but also an indicator of other pathogens being present. Therefore, coliform is a significant marker as it helps raise awareness and determine the source of other pathogenic bacteria. Based on this approach, we isolated coliform bacteria from twelve fruit juice samples (local and packed) available in Rajshahi City. Among these samples, a lactose non-fermenting white-colored coliform colony (Isolate A) from sugarcane juice (Sample 6) and lactose fermenting pink-coloured colony (Isolate B) from packed mango drinks (Sample 9) were chosen for further research based on colony number and morphology. Morphological features are key to determining the general characteristics of bacteria. The first report by Kabler and Clark (Kabler and Clark 1960 ), showed that coliforms were rod-shaped, gram-negative, non-spore-forming bacteria that ferment lactose within 24 hours at a temperature of 37°C. Our findings also proved that the isolated bacteria were gram-negative, non-motile, and rod-shaped. Maximal growth was observed at different pH levels and temperatures. In this instance, isolate A exhibited the highest growth at a pH of 7.0 and a temperature of 35°C, whereas isolate B displayed maximum growth at a pH of 7.2 and a temperature of 37°C. According to Bergey’s Manual of Systematic Bacteriology (Garrity, 2006 ), the growth temperature and pH range for Acinetobacter sp. is 35°-37°C and 7.0-7.5, respectively. Additionally, Bergey's Manual of Determinative Bacteriology (Breed et al., 1957 ) indicates that the pH range for Klebsiella sp. is between 7.0 and 7.8, while the growth temperature range is from 30° to 37°C. Therefore, it is confirmed that the physiological and characteristic outputs of the isolated bacteria had a resemble the previous findings. Biochemical tests are among the principal methods for microbial identification (Atlas of Oral Microbiology 2015). Bergey’s Manual of Systemic Bacteriology (Garrity, 2006 ), confirmed the morphological and biochemical test results of the isolates as members of the genus Acinetobacter and Klebsiella , respectively. The 16S rRNA gene sequencing is regarded as a favored genetic method for identification at the molecular level (Clarridge et al. 2004). Molecular identification indicated that bacterial isolate A had a 97% significant alignment with Acinetobacter sp. (Accession no. LC437022), whereas isolate B had a 98% significant alignment with Klebsiella sp. (Accession no. LC552682). Asif et al. reported Acinetobacter baumannii as a persistent infectious agent (Asif et al., 2018 ), while Martin and Bachman classified Klebsiella pneumoniae as an opportunistic, hypervirulent, and multidrug-resistant pathogen based on a genome study (Martin and Bachman, 2018 ). Neves et al. assessed acute dinoflagellate toxicity at a concentration of 200 cells ml⁻¹, which affected the germination and survival rates of Artemia salina during the stationary and exponential phases, respectively (Neves et al. 2017 ). They also observed the highest tD 50 value for G . excentricus within 4 hours in the case of the exponential phase. In our present study, we evaluated the toxicity of isolated bacteria, and developmental abnormalities were also identified in both the stationary and exponential phases of Artemia salina at 200 cells ml − 1 . Arulvasu et al. reported the impact of silver nanoparticles on both alive animals and cysts. In their findings, brine shrimp guts filled with nanoparticles showed a significant mortality level within 24 hours of exposure (Arulvasu et al., 2014 ). Pinheiro et al. reported the accumulation of MTiO 2 in Artemia sp. after 24 hours and 48 hours without any morphological damage observed in the control group (Pinheiro et al. 2023 ). In their experiment, MTiO 2 was found in the gut of Artemia sp., and the cephalothorax showed swelling at concentrations of 50 ppm and 100 ppm, respectively. Additionally, the positive control K 2 Cr 2 O 7 exhibited underdeveloped body growth within 24 hours of exposure. However, MTiO 2 dispersed throughout the body of Artemia sp. resulting in morphological changes such as tissue degradation at the highest concentration of 100 ppm, while individuals exposed to K 2 Cr 2 O 7 showed improper development with deformities in the appendages and abdomen. In the present study, during the stationary phase, isolated bacteria had a toxic effect on germination of Artemia salina as both isolates showed delayed germination after 24 hours, while the control group showed germination after 16 hours. The variation between the treatment and control group was significant. In the case of the stationary phase, damaged germinated nauplii were found after 24 hours of Acinetobacter sp. treatment, exhibiting missing antenulla, abnormal eyes, contused mandibles, absent swimming legs, and disrupted structure. After 48 hours of treatment with Acinetobacter sp. missing antenulla, absent swimming legs, and damaged ovaries were observed. Similarly, germinated deformed nauplii were observed after 24 hours of treatment with Klebsiella sp., while missing antenulla, absent swimming legs, and damaged ovaries were observed after 48 hours. It was reported that Klebsiella pneumoniae caused mortality in Nile tilapia (Silva et al. 2021). Bi et al. described disease-causing Acinetobacter johnsonii in Rainbow Trout (Bi et al. 2023 ). Again, during the exponential phase, the treatment with Acinetobacter sp. exhibited the highest level of toxicity. After 8 hours, it showed no survival with significant variation compared to the control group. Developmental abnormalities such as abnormal eye, improperly developed mandibles, absent swimming legs, defective structure, and abnormal telsion width were also identified after 8 hours of treatment. Moreover, Artemia treated with Klebsiella sp. exhibited low survival after 8 hours compared to previous hour, and the variation was significant. Developmental abnormalities missing antennula, deformed swimming legs, incorporeal ovary, and abnormal telsion width were also observed. In addition, the tD 50 was found to be within 5 hours for Acinetobacter sp. and 6 hours for Klebsiella sp., in the exponential phase. Similar abnormalities were found in a previous study by Talukder et al (Talukder et al. 2019 ), and our findings aligned with their data. Therefore, it is evident that the toxicity level of Acinetobacter sp. was higher than that of Klebsiella sp. in both phases of Artemia salina . Overall, the toxicity of isolated coliforms at the developmental level was significant and analyzed through DMRT. Furthermore, the ANOSIM result confirmed significant changes compared to the control group in both the stationary (R = 0.646; P < 0.001) phase and exponential phase (R = 0.52; P < 0.002). Moreover, the Pearson correlation validated the negative correlation between the control group and the treated Artemia in both cases. Brine shrimp bioassay is also used for cytotoxicity tests of microbial toxins and bioactive compounds. We also tested the toxicity of the isolates against Artemia salina and the LC 50 values for Acinetobacter sp. and Klebsiella sp. were 61.07 ± 0.19 µl and 69.99 ± 0.21 µl, respectively after 24 hours of exposure. From the results, it was clear that Acinetobacter sp. exhibited higher toxicity than Klebsiella sp. against aquatic organisms. We identified several multidrug-resistant (MDR) isolates in the current investigation. Our results showed that Acinetobacter sp. (Isolate A) was resistant to tetracycline, ciprofloxacin, penicillin, ceftazidime, cefixime, and doxycycline, while susceptible to kanamycin, gentamycin, erythromycin, amoxicillin, and cefuroxime, and had intermediate resistance only against ampicillin. In hospitalized patients, Dortet et al. isolated Acinetobacter ursingii and Acinetobacter schindleri that were resistant to cefixime and susceptible to amoxicillin, kanamycin, and gentamycin (Dortet et al. 2006 ). The previous report found multidrug-resistant Acinetobacter sp. from fruits, which exhibited 60% resistance against ceftazidime and 84% resistance against ciprofloxacin (Carvalheira, 2017). Another investigation carried out by Pal et al. reported that Acinetobacter baumannii was 100% resistant against penicillin (Pal et al. 2017 ), followed by Ferdous et al. who showed a resistance pattern of Acinetobacter sp. isolated from the hospital exhibiting 68.5% resistance against tetracycline, 92% resistance against ampicillin, and 84.9% resistance against ciprofloxacin (Ferdous et al. 2016 ). So, it is confirmed that our results are in accordance with previous findings. On the other hand, our results depicted that Klebsiella sp. (Isolate B) was resistant against penicillin, tetracycline, doxycycline, and cefixime, intermediately resistant against ampicillin, and erythromycin, but susceptible to amoxicillin, kanamycin, ceftazidime, gentamycin, ciprofloxacin, and cefuroxime. According to Khalif et al. an antibiogram study of pathogenic bacteria from street-vented food showed that Klebsiella sp. was susceptible to kanamycin, gentamycin, and ciprofloxacin, but resistant to erythromycin (Khalif et al. 2018 ). Another study revealed that multidrug-resistant Klebsiella pneumonae isolated from powdered infant formula showed 15.4% resistance against cefixime, 53.8% resistance against ampicillin, 38.5% susceptibility to cefuroxime, 84.6% susceptibility to ceftazidime, and 100% susceptibility to ciprofloxacin, gentamycin, and kanamycin (Zhou et. al. 2011 ). In addition, Padamadan et al. (Padamadan et al. 2016 ) reported an antibiogram of street-vended and packed fruit juices in which Klebsiella sp. was resistant against ampicillin, cefixime, and penicillin. Hence, it can be concluded that our research output had similarities with the referred data. From the results, it is clear that Acinetobacter sp. was more resistant to antibiotics in contrast to Klebsiella sp. So, both screened bacteria might be a matter of health issues without proper consciousness as well as without assuring the hygienic status of juice during processing, storage, selling, and drinking by children or people. 5. Conclusions From the results of the current study, it has been confirmed that fruit juices (both street-vented and packaged) show higher levels of contamination with pathogens, including Acinetobacter sp. and Klebsiella sp. Additionally, these indicative pathogens have shown resistance to multiple antibiotics. The DMRT analysis revealed notable abnormalities in Artemia salina due to multidrug-resistant coliforms in terms of coliform developmental toxicity. Furthermore, ANOSIM and ordination MDS indicated significant abnormalities among bacteria-treated Artemia compared to the control group. Pearson correlation analysis showed that, longer exposure times of coliforms to Artemia salina were significantly positively correlated with abnormalities when compared to the control group. Since fruit juices are popular beverage, their quality is of utmost importance to human health. The microbial contamination of these juices poses a serious threat as it continues to be a significant source of illness, particularly among children, and affect the health of population in both developed and developing regions. Therefore, storage strategies should be implemented to ensure the microbial standards of street-vented and commercially available fruit juices, in order to guarantee food security and safety. This way, the spread of antibiotic-resistant infections within the population can be prevented. Declarations Ethical Approval: Ethical approval was not applicable to this research as it did not involve human participants, animals, or related ethical concerns. Informed Consent Statement: Not applicable Data Availability Statement: All data can be obtained from the corresponding authors upon reasonable request. Patents: Not applicable Funding: This work received no external funding from any sources. Declaration of Consent to Participate: This study did not involve human participants; therefore, a 'Consent to Participate' declaration is not applicable. Author Contributions: Conceptualization, M.N.H., and M.A.E.; Methodology, M.N.H., M.A.E., and A.P.T.; Data analysis, M.A.E., and M.N.H.; Data curation, M.N.H., M.A.E., A.P.T., and S.S.D.; Writing-original draft, M.N.H., A.P.T., and S.S.D.; Writing-reviewing and editing, M.A. S., M.A. E.; Visualization, M.A.S. and S.Z.; Supervision, M.S.U., and M.A.E.; Laboratory facilities, M.A.E., M.A.S., M.S.U., and S.Z. All of the authors read and approved the manuscript. C onflicts of Interest: The authors declare no conflicts of interest. References Abbo, M.; Olurin, T.O.; Odeyemi, G. 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A microwell cytotoxicity assay using Artemia salina (brine shrimp). Planta Med. 1993 , 59 , 250-252. Suaads, A.; Hamed, E. Microbial growth and chemical analysis of bottled fruit juices and drinks in Riyadh, Saudi arabia. Res. J. Microbiol. 2008 , 7 , 315-325. Talukder, A.P.; Haque, M.N.; Zaman, R.; Ekram, M.A.E. Profiling resistance pattern and assessing toxicity of coliform bacteria on the developmental stages of Artemia salina isolated from drinking water of northern part of Bangladesh. Indian. J. Microbiol. Res. 2019 , 6 , 355-366. Tasnim, F.; Hossain, M.A.; Nusrath, S.; Hossain, M.K.; Lopa, D.; Haque, K.M.F. Quality assessment of industrially processed fruit juices available in Dhaka city, Bangladesh. Malays. J. Nutr. 2010 , 16 , 431-438. Zhou, X.; Gao, J.; Huang, Y.; Fu, S.; Chen, H. Antibiotic resistance pattern of Klebsiella pneumoniae and Enterobacter sakazakii isolates from powdered infant formula. Afr. J. Microbiol. Res. 2011 , 5 , 3073-3077. Zhu, B.; Zhu, S.; Li, J; Hui, X.; Wang, G.X. (2018). The developmental toxicity, bioaccumulation and distribution of oxidized single walled carbon nanotubes in Artemia salina . Toxicol. Res. 2018 , 7 , 897-906. Additional Declarations No competing interests reported. Supplementary Files SupplimentaryFile.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6737003","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":464455473,"identity":"3e702e0d-f3ab-4453-bfec-82084dad6930","order_by":0,"name":"Md. 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Abu Saleh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYFAC5gZmhgook4GNKC2MQC1nSNbC2EaKFvn2g42PC+fdS2xgP/yAuaCMCC0GZxKbjWduK05s4EkzYJ5xjhgtDIlt0rzbEhIbGHIYmHnbiHFY/8P237xzgFr43xCpheFGYhszbwNQiwSxthjceNgszXMswbhN4pnBYaL8It+ffPAzT02CbD9/8sPHRIUYHIBi5AApGkbBKBgFo2AU4AEAwv8x1LAP+HUAAAAASUVORK5CYII=","orcid":"","institution":"University of Rajshahi","correspondingAuthor":true,"prefix":"","firstName":"Md.","middleName":"Abu","lastName":"Saleh","suffix":""},{"id":464455478,"identity":"02f11a81-7ed2-4aca-9dfc-a4806d8c6047","order_by":5,"name":"Shahriar Zaman","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Shahriar","middleName":"","lastName":"Zaman","suffix":""},{"id":464455479,"identity":"83428cb0-9616-4bcf-b5c4-000dbed31058","order_by":6,"name":"Md. Akhtar-E-Ekram","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"","lastName":"Akhtar-E-Ekram","suffix":""}],"badges":[],"createdAt":"2025-05-24 05:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6737003/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6737003/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83817865,"identity":"d6a2026f-986e-4fdc-99d0-156b166ad2cc","added_by":"auto","created_at":"2025-06-03 08:05:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39409,"visible":true,"origin":"","legend":"\u003cp\u003eThe presence of coliform bacteria in twelve different samples (local street juice = S1 to S7 and packed juice = S8 to S12) isolated from different packed and local juices. All samples were screened in triplicate using the MacConkey medium to count the coliform CFU numbers.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6737003/v1/7007fdbc4e812f66fca50832.png"},{"id":83817866,"identity":"dcf39d9a-27ec-4891-82a6-1ac5f1d91087","added_by":"auto","created_at":"2025-06-03 08:05:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31992,"visible":true,"origin":"","legend":"\u003cp\u003eThe graph indicates the variation in the germination number of Artemia salina during the stationary phase through DMRT analysis\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6737003/v1/11fc1c4db73adf80767f2fb6.png"},{"id":83817867,"identity":"aceeb3e5-cabd-4281-9226-9b206d60089a","added_by":"auto","created_at":"2025-06-03 08:05:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":355790,"visible":true,"origin":"","legend":"\u003cp\u003eThe toxic impacts of isolated bacteria \u003cem\u003eAcinetobacter \u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e (A) and \u003cem\u003eKlebsiella\u003c/em\u003esp. (B) on the stationary phase of\u003cem\u003e Artemia salina\u003c/em\u003e. The stationary phase denotes the treatment of the coliform bacteria prior to the germination of cysts in order to observe defects in the germinated nauplii, such as damaged nauplii in the case of \u003cem\u003eAcinetobacter \u003c/em\u003esp. and deformed nauplii observed in the case of \u003cem\u003eKlebsiella\u003c/em\u003esp.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6737003/v1/8e7b9b6a3edbd2d01808ef22.png"},{"id":83817868,"identity":"64353896-aa76-4494-aa88-4a0eb0f51552","added_by":"auto","created_at":"2025-06-03 08:05:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40296,"visible":true,"origin":"","legend":"\u003cp\u003eThe bar graph shows the variation of \u003cem\u003eArtemia salina \u003c/em\u003esurvival rate after treatment with the isolated two bacteria and the control without treatment through DMRT at the exponential phase. All data were recorded in triplicates.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6737003/v1/170a309cd9aa830a91cb18a3.png"},{"id":83817871,"identity":"63d48127-b3f2-4874-8f1c-a84dc1fdcd70","added_by":"auto","created_at":"2025-06-03 08:05:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":464119,"visible":true,"origin":"","legend":"\u003cp\u003eThe toxic impacts of isolated bacteria \u003cem\u003eAcinetobacter \u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e (A) and \u003cem\u003eKlebsiella\u003c/em\u003esp. (B) on the exponential phase of\u003cem\u003e Artemia salina\u003c/em\u003e. The exponential phase indicates the bacterial treatment after the germination of cysts to observe and count the defects in the developmental nauplii, such as the defective structure of mandibles, eyes, swimming legs, and telson in the case of both \u003cem\u003eAcinetobacter \u003c/em\u003esp. and also in \u003cem\u003eKlebsiella\u003c/em\u003e sp.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6737003/v1/d520b6f9bedd2645f6fb9a5c.png"},{"id":83817869,"identity":"e402ec30-0e4c-4866-b67f-474910ed05bd","added_by":"auto","created_at":"2025-06-03 08:05:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":338180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a). \u003c/strong\u003eHeat map shows the significant Pearson correlation among abnormal traits of \u003cem\u003eArtemia salina\u003c/em\u003e at 24 hr, 36 hr, and 48 hr, and the dose of bacteria \u003cem\u003eAcinetobacter \u003c/em\u003esp. and \u003cem\u003eKlebsiella \u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e The isolated bacteria were used to treat the \u003cem\u003eArtemia\u003c/em\u003ecysts, and the results were recorded in triplicate for correlation (stationary phase) and compared to the control. Here, CM= Contused mandible, AE=Abnormal eye, MA= Missing antennula, ASL= Absent swimming legs, DS=Disrupted structure, GDN= Germinated damage nauplii, DO= Damaged ovary, SA= Smashed area. The yellow color key signifies a higher R-value, whereas the blue denotes a lower value.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b). \u003c/strong\u003eHeat map shows the significant Pearson correlation among abnormal traits of \u003cem\u003eArtemia salina\u003c/em\u003e at 5 hr, 36 hr, 7 hr, 8hr and the dose of bacteria \u003cem\u003eAcinetobacter \u003c/em\u003esp. and \u003cem\u003eKlebsiella\u003c/em\u003esp. The germinated cysts were treated with bacteria and developments were recorded until 8hr after treatments (exponential phase). Here, IO= Incorporeal ovary, IDM=Improperly developed mandibles, ATW= Abnormal telson width, DSL= Deformed swimming legs. The yellow color key signifies a higher R-value, whereas the blue denotes a lower value.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6737003/v1/1d61388f7959d47affc86c21.png"},{"id":83817872,"identity":"2e484478-6ed9-4926-a6f1-b969d08bcfb8","added_by":"auto","created_at":"2025-06-03 08:05:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":150542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eMultidimensional scaling (MDS) ordination for the stationary phase abnormalities dataset, grouped by color based on the three different hour-based observations and records. The germinated deformities of \u003cem\u003eArtemia\u003c/em\u003e were counted after 24 hours, 36 hours, and 48 hours. The control without treatment was shown in blue color-coded here.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. \u003c/strong\u003eMultidimensional scaling (MDS) ordination for the exponential phase abnormalities dataset and grouped by color-coded based on the four different hour-based observations and records. The developmental deformities of \u003cem\u003eArtemia \u003c/em\u003ewere counted after 5 hours, 6 hours, 7 hours, and 8 hours. The control without treatment was shown in blue color-coded here.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6737003/v1/020942a85f7cb047484e42e6.png"},{"id":83818709,"identity":"3b9385b2-9db6-4657-8e80-475cf711debb","added_by":"auto","created_at":"2025-06-03 08:14:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2789099,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6737003/v1/9f53fd9f-8ece-4989-a0d9-a1f5b7a7384c.pdf"},{"id":83817870,"identity":"5eaa4175-0b8a-4c7b-b987-05c10e91abf4","added_by":"auto","created_at":"2025-06-03 08:05:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":719462,"visible":true,"origin":"","legend":"","description":"","filename":"SupplimentaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-6737003/v1/43e4f0be288e6cf018af7ab7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening of multidrug-resistant coliforms from fruit juice and their significant correlations with the developmental deformities of Artemia salina (Brine shrimp)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFruits are an essential ingredient of a well-balanced aliment and fruit juices are widely consumed as a source of essential minerals, nutrients, antioxidants, and dietary fibres. Fruit juices are nutrient-dense beverages that taste great and have several health advantages (Suaads and Hamed \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Juice contains significant quantities of vitamins C and E along with antioxidants, and has a nice flavour and aroma. These essential restorative properties may reduce the risk of many diseases (Abbo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Notably, fruit juices play immense roles in human body detoxification, prevention of vitamin A and vitamin C deficiencies as well as enhancement in the blood lipid profile of individuals with hypercholesterolemia. Regular intake of fruit juice is linked with reducing the chances of developing cataracts, Alzheimer's disease, cancer, heart disease, stroke, as well as other anomalies associated with aging (Liu \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). For instance, Mango juice contains components like phytochemicals and antioxidants (Liu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which are very helpful for curing serious ailments as well as cancer. A glass of chilled sugarcane juice not only satisfies our thirst but also rejuvenates us.\u003c/p\u003e \u003cp\u003eNevertheless, even if fruit juice has a high nutritional value, the production and processing practices in developing countries may not reach suitable safety levels and pose consumers at risk of illness. Foodborne diseases are one of the major reasons for global fatalities, predominantly in developing countries (Oladipo \u0026amp; Adejumobi \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Several studies reported food-borne illness caused by the consumption of fruit juices and the prevalence of coliforms on fruit surfaces is a clear indication of fecal pollution which can be detrimental to human health (Reddy et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Padamadan et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A wide range of diverse microorganisms known as \"coliform bacteria\" that are utilized as indicator organisms for assuring the presence of pathogenic contagious microbes, especially bacteria in foods and water. The key characteristics of coliform include its aerobic nature, gram-negative status, lack of spore formation, rod-like morphology, and lactose fermentation (Isfahani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Juice processing with unhygienic water can be a most important vehicle for microbial shipment, for instance fecal coliforms, coliforms as well as fecal \u003cem\u003eStreptococci\u003c/em\u003e etc. (Tasnim et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Juices are latent sources of harmful bacteria including \u003cem\u003eStaphylococcus aureus, Salmonella\u003c/em\u003e sp., \u003cem\u003eE. coli\u003c/em\u003e 0157, \u003cem\u003eShigella\u003c/em\u003e sp., \u003cem\u003eErwinia\u003c/em\u003e sp., \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e, \u003cem\u003eEnterobacter\u003c/em\u003e sp., \u003cem\u003eAcinetobacter\u003c/em\u003e sp., \u003cem\u003ePseudomonas\u003c/em\u003e sp., and \u003cem\u003eAlicyclobacillus\u003c/em\u003e sp. (Lewis et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). For instance, \u003cem\u003eAcinetobacter\u003c/em\u003e sp. is frequently responsible for illnesses including meningitis, skin and soft tissue infections, bacteremia, respiratory and urinary tract diseases (Oberoi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Furthermore, the most prevalent Enterobacteriaceae pathogenic bacteria that cause nosocomial infection outbreaks is \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, which is especially common in critical care, burn, oncology, and neonatal units (Correa et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Prior research has revealed that fruit juices from street vendors as well as those in packs are contaminated with harmful microbes (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Bangladesh, street-vented and packed fruit juices are popular among people because they fulfill nutritional requirements at a reasonable price. Nonetheless, the easy availability of fruit juices is a familiar source of pathogenic bacterial transmission in developing countries like Bangladesh (Sarker et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Das et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). There are several reasons behind this microbial contamination. Firstly, lack of personal hygiene, conventional processes of juice preparation, and inappropriate preserving temperature are the most important factors (Rane \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Additional justifications include fruit flies, filth, and improperly cleaned tools like cutters and knives, as well as direct contamination from rotting fruits (Reddy et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Because of improper handling and serving procedures, street-vended fruit juices are frequently linked to diarrheal illnesses in Bangladesh (Bello et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Due to these negative consequences of consuming contaminated fruit juices, it is indispensable to measure the coliform\u0026rsquo;s acute toxicity in a model organism to address the microbial health hazards risk from fruit juice.\u003c/p\u003e \u003cp\u003e \u003cem\u003eArtemia salina\u003c/em\u003e (brine shrimp) is a widely recognized crustacean found in seawater environments (saline lakes and seas) and a vital food source for fish and other aquatic invertebrates (Zhu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The model organism has been widely used to identify the toxic effect of bacteria, chemicals due to its year-round commercially availability of dried cysts, low cost, short life cycle and easy to maintain in lab at in vivo conditions (Faimali et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This assay is regarded as a reliable way to assess the level of toxicity in plant extract (Solis et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), and evaluation of toxic cyanobacteria (Lahti et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and heavy metals (Ates et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Previous studies revealed a high association between the cytotoxicity directed against certain tumor cell lines and the toxic activities in the brine shrimp experiment (Anderson et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) as well as the hepatotoxic activity (Kiviranta et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Thus, \u003cem\u003eArtemia salina\u003c/em\u003e experiment is a more feasible way compared to cell lines assays for in vivo acute toxicity evaluation (Piccardi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, our research aimed to investigate coliform bacterial availability in collected fruit juice (locally and commercially) and coliform\u0026rsquo;s acute toxicity detection through \u003cem\u003eArtemia Salina\u003c/em\u003e. The aim also includes a statistical justification of significant correlation between coliform and coliform-treated abnormalities of \u003cem\u003eArtemia\u003c/em\u003e (during stationary and exponential phase) and their antibiotic-resistant status to understand the possible public health concern.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sample Collection\u003c/h2\u003e \u003cp\u003eTwelve samples of fruit juice were taken from different areas of the Rajshahi metropolitan area (site-1: Laxmipur, site-2: Railway station, site-3: University campus, and site-4: Shaheb bazar) in May 2022. Among the twelve samples, Sample 1 to Sample 7 were street-vended fruit juice samples, namely papaya juice for Sample 1, orange juice for Sample 2, lemon juice for Sample 3, bael juice for Sample 4, watermelon juice for Sample 5, sugarcane juice for Sample 6, and pineapple juice for Sample 7. The remaining five samples were packed mango juices samples identified as Sample 8 to Sample 12). Subsequently, all of the samples were taken directly to the Microbiology Laboratory of the Department of Genetic Engineering and Biotechnology at the University of Rajshahi to evaluate their microbiological quality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Isolation of Coliform Bacteria and Optimization of Their Growth Parameters\u003c/h2\u003e \u003cp\u003eThe samples of fruit juices were collected as a source of inoculum, and droplets of juice were used for plating on MacConkey agar medium, which contains the following substances: (Lactose monohydrate 10.0 g/L, peptones 3.0 g/L, bile salts 1.5 g/L, sodium chloride 5.0 g/L, crystal violet 0.001 g/L, and Agar 13.5 g/L at pH\u0026thinsp;=\u0026thinsp;7.0) separately. The plates were subsequently incubated at 37\u0026deg;C for a duration of 24 hours. Two coliform bacteria were selected and marked as A and B based on lactose fermentation capacity and healthy colony morphology. These two bacteria showed highest CFU (colony forming unit) among all other bacteria which were 8.5\u0026times;10\u003csup\u003e9\u003c/sup\u003e cfu/ml and 6.8\u0026times;10\u003csup\u003e7\u003c/sup\u003e cfu/ml for Isolate A and Isolate B, respectively. Lastly, strains that had been isolated as pure cultures were kept for use in future research.\u003c/p\u003e \u003cp\u003eDifferent temperatures and pH influence bacterial growth. In this experiment, the pH was varied at 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0, while the temperature was 25, 30, 35, and 40\u0026deg;C. In both cases, the optical density of the bacterial liquid cultures was recorded with a double-beam spectrophotometer (Analytk Jena AG, Germany), at 660 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Morphological and Biochemical Characterization of Isolated Bacteria\u003c/h2\u003e \u003cp\u003eNumerous morphological techniques, such as Gram staining and motility, as well as biochemical assays, including methyl red, catalase, urea hydrolysis, Simmons citrate, and TSI test, were used to characterize the isolates.\u003c/p\u003e \u003cp\u003eTo differentiate between gram-negative and gram-positive bacteria by analysing their cell wall structures, gram staining (0.5% Crystal violet solution, 1% Lugol's iodine solution, and 1% safranin solution) was conducted. Motility test (Agar 4.0 g/L, NaCl 5.0 g/L, and Peptone 10.0 g/L) was used to observe bacterial planktonic status. Among the biochemical test, catalase test (0.5 g/L of yeast extract, 1 g/L of peptone, and 2.4 g/L of agar) was performed to check hydrogen peroxide detoxification ability of aerobic bacteria. Methyl red test (Basal salt media: 2.34g of K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, 1.33g of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.02g of MgSO\u003csub\u003e4\u003c/sub\u003e. 7H\u003csub\u003e2\u003c/sub\u003eO, 1.00g of (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 0.50g of NaCl, 0.10g of yeast extract, and 1.00g of glucose) was used to know the ability of microorganism\u0026rsquo;s mixed acid fermentation. Along with these tests, the citrate test (1.05 g/L of bromothymol blue, 1.0 g/L of ammonium dihydrogen phosphate, 1.0 g/L of dipotassium phosphate, 2.0 g/L of sodium citrate dehydrate, 5.0 g/L of sodium chloride, 15.0 g/L of agar, and 0.20 g/L of magnesium sulphate heptahydrate) was done for citrate utilizing ability, urease test (Agar 15.0 g/L, 0.012 g/L of phenol red, 1.0 g/L of dextrose, 1.20 g/L of disodium phosphate, 0.80 g/L of monopotassium phosphate, 5.0 g/L of sodium chloride, and 1.0 g/L of peptic digest of animal tissue) for urease enzymatic activity, and the triple sugar iron agar test was used to determine whether lactose, sucrose, and dextrose could ferment. The morphological and biochemical tests were conducted as per standard protocols (Collin et al.1995).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Molecular Identification of Isolated Bacteria\u003c/h2\u003e \u003cp\u003eThe following procedures were used to identify the isolated bacteria at the molecular level: Extracting the chromosomal DNA, amplifying the 16S rRNA gene, purifying the PCR product, cycle sequencing with purifying the cycle sequencing product, detecting the nucleotides, and sequence analysis.\u003c/p\u003e \u003cp\u003eThe CTAB technique was used to extract the bacterial genomic DNA described by Edwards et al. (Edwards et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). After extraction, The NanoDrop Spectrophotometer (Thermo Scientific, USA; ND2000 series) was used to quantify DNA. PCR was used to amplify the 16S rRNA gene following the instructions provided by Loffler et al. (Loffler et al. 2000) using 16s F and 16s R (Sigma, USA) universal primers. The primer sequences are provided in the supplementary file (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All PCR reagents were used from the Promega (Catalogue no. M7432, Promega, USA). The composition of the PCR Reaction mix (Total volume is 25 \u0026micro;l) are in supplementary (Table S2). The reagents were mixed gently, and short spin was done. PCR amplification was done by Gene Atlas (G2; Astec, Japan). Cycling parameters for PCR analysis in supplementary. After PCR amplification, samples were run in agarose gel (Catalogue no. V3125; Promega, USA) for visualization of the band. In this process, Promega, USA's 1 kb DNA ladder (Catalogue no. G5711), TAE buffer (Catalogue no. V4251), and ethidium bromide solution (Catalogue no. H5041) were utilized. In order to purify the PCR product, 40 \u0026micro;l of the product was combined with 160 \u0026micro;l of sterile water, resulting in a final amount of 200 \u0026micro;l. Following that, the 200 \u0026micro;l was transferred to the YM-100 rapid spin columns and centrifuged for 10 minutes at 500 xg. The columns were moved into fresh tubes for the DNA elution process, and added 25 \u0026micro;l of sterile water. Next, for five minutes, the tubes were centrifuged at 1000 xg. Genetic Analyzer (3500, Thermo Scientific, USA) was used for the sequencing of the PCR-purified product. After that, the bioinformatics tool Chromas was used to edit the sequences. Then the BLASTN algorithm (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to determine the homology between the 16S rRNA gene sequence and the gene sequences of other organisms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Evaluating the Toxic Effects of Isolated Bacteria on the Developmental Stages of Artemia salina\u003c/h2\u003e \u003cp\u003eAn acute toxicity assay was conducted to compare the cellular toxicity of coliform bacterial strains obtained from fruit juice at two developmental stages of \u003cem\u003eArtemia salina\u003c/em\u003e: the stationary and the exponential phases.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Stationary Phase\u003c/h2\u003e \u003cp\u003eAssays for intoxication were conducted using 6-well plates, with 10 ml of filtered seawater in each well. Ten \u003cem\u003eArtemia salina\u003c/em\u003e cysts were co-cultured and incubated with isolated bacteria that had grown overnight at an optical density of 0.5, with three replications at an abundance of 200 cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A control containing brine shrimp without isolated bacteria was also implemented in three times. In this case, a mineral salt (MS) liquid medium was used for bacterial culture. Every six hours during the 48-hour incubation period, the germination of the cysts was inspected, and any significant anomalies were noted using an inverted microscope (USA: LABOMED CXL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Exponential Phase\u003c/h2\u003e \u003cp\u003eThe experiment was performed using 6-well plates, with each well containing 10 ml of filtered seawater, and this phase was observed for up to 7 hours. The experiment was carried out in triplicate with a control group and involved incubating six individuals of \u003cem\u003eArtemia salina\u003c/em\u003e for seven hours with overnight cultured isolated bacteria (O.D. at 0.5) at an abundance of 200 cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Every hour after incubation, the survival of individuals and significant anomalies were examined under an inverted microscope (LABOMED CXL, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Determination of tD\u003csub\u003e50\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eIn the case of the exponential phase, the tD\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e (the time when 50% \u003cem\u003eof Artemia salina\u003c/em\u003e died) value was calculated following the protocol outlined by Neves et al. (Neves et al.2017) [30]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Determination of LC\u003csub\u003e50\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eThe lethality bioassay of brine shrimp is considered a helpful method for conducting an initial toxicity assessment (Solis et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Nauplii were obtained by hatching \u003cem\u003eArtemia salina\u003c/em\u003e eggs in simulated seawater. To evaluate the cytotoxic activity of the isolated bacteria on brine shrimp, six autoclaved test tubes were prepared, and 10 ml of artificial seawater containing 10 nauplii were introduced into each tube. Finally, 25, 50, 75, 100, 125, and 150 \u0026micro;l of the previously cultured bacterial samples were sequentially added to the test tubes using a micropipette. The test tubes exposed to light and incubated at ambient temperature for 24 hours to monitor the cytotoxic effects. After the 24-hour period, the number of survivors was recorded. This data was then analyzed using a basic probit analysis tool to calculate the LC\u003csub\u003e50\u003c/sub\u003e value with 95% confidence intervals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis for significant changes and correlation between coliform doses and Artemia abnormality\u003c/h2\u003e \u003cp\u003eFor statistical analysis, the Duncan Multiple Range Test (DMRT) was conducted using Statistical Analysis Software (SAS, version 9.1.3) to compare the means of three replicates between groups. To visualize the significant changes through MDS, the data of Artemia cysts treated with coliform bacteria Isolate A and isolate B were normalized and analyzed separately for the stationary and exponential phases. ANOSIM (Analysis of Similarity) was performed to determine significant differences in abnormalities between coliform-treated \u003cem\u003eArtemia\u003c/em\u003e and untreated \u003cem\u003eArtemia\u003c/em\u003e using Primer E (Version 7). To measure the Pearson correlations between the abnormal features of treated \u003cem\u003eArtemia\u003c/em\u003e and the duration of dose, heatmaps were generated using the heatmap package in R (version 4.1.3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Antibiotic Sensitivity Test\u003c/h2\u003e \u003cp\u003eThe antibiotic sensitivity profile of isolated coliform bacteria was assessed using the Kirby-Bauer disc diffusion technique (Bauer et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). In this study, both Luria broth (LB) liquid and LB agar were utilized as culture media to facilitate bacterial growth. Commercially available and commonly recommended antibiotics such as penicillin (10 \u0026micro;g per disc), amoxicillin (10 \u0026micro;g per disc), erythromycin (15 \u0026micro;g per disc), ampicillin (10 \u0026micro;g per disc), kanamycin (30 \u0026micro;g per disc), tetracycline (30 \u0026micro;g per disc), ceftazidime (30 \u0026micro;g per disc), gentamycin (10 \u0026micro;g per disc), ciprofloxacin (5 \u0026micro;g per disc), cefuroxime (30 \u0026micro;g per disc), cefixime (5 \u0026micro;g per disc), Doxycycline (10 \u0026micro;g per disc) were used as antibiotic discs, placed in the center of the corresponding plates, and incubated overnight at 37\u0026deg;C. Following an overnight incubation period, the zones were examined and measured using the mm scale.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e3.1. Isolation and Optimization of Growth Characteristics of Isolated Bacteria\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eColiform bacterial strains were isolated from the collected fruit juices through screening on MacConkey agar media and the highest number of colonies were found in the following pattern: Sample 6\u0026thinsp;\u0026gt;\u0026thinsp;Sample 9\u0026thinsp;\u0026gt;\u0026thinsp;Sample 4\u0026thinsp;\u0026gt;\u0026thinsp;Sample 2\u0026thinsp;\u0026gt;\u0026thinsp;Sample 1\u0026thinsp;\u0026gt;\u0026thinsp;Sample 12\u0026thinsp;\u0026gt;\u0026thinsp;Sample 7\u0026thinsp;\u0026gt;\u0026thinsp;Sample 5\u0026thinsp;\u0026gt;\u0026thinsp;Sample 10\u0026thinsp;\u0026gt;\u0026thinsp;Sample 3 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Out of the twelve samples, Sample 6 (street juice with a white colony) had the highest number of coliform colonies, and Sample 9 (packed juice screened by a pink colony) was chosen for additional tests. However, Samples 8 and 11, both packed juices, showed no growth on MacConkey agar media (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eAccording to our findings, the maximum growth of bacteria was observed in isolates A (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) and B (Figure S2) at pH 7.0 and 7.2, respectively. The optimal temperature for bacterial growth was 35\u0026deg;C for isolate A (Figure S3) and 37\u0026deg;C for isolate B (Figure S4).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Molecular Profiling of Isolated Bacteria\u003c/h2\u003e\n \u003cp\u003eFollowing 16S rRNA gene sequencing and editing, the sequences were compared to those of other organisms that had previously been uploaded to the GenBank database Some sequences with notable identities were found in the Gen Bank, and isolate A showed a significant 97% similarity with \u003cem\u003eAcinetobacter\u003c/em\u003e sp. (Accession no. LC437022), while isolate B showed a 98% similarity with \u003cem\u003eKlebsiella\u003c/em\u003e sp. (Accession no. LC552682). The PCR band of the chromosomal DNA and the DNA quantification analysis are shown in Figs. S5-S7, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Evaluating the Toxicity of Isolated Bacteria on the Developmental Stages of Artemia salina\u003c/h2\u003e\n \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1. Stationary Phase\u003c/h2\u003e\n \u003cp\u003eAfter 16 hours, \u003cem\u003eArtemia\u003c/em\u003e cysts treated with both isolates (\u003cem\u003eAcinetobacter\u003c/em\u003e sp. and \u003cem\u003eKlebsiella\u003c/em\u003e sp.) showed no germination (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Next to 24 hours, both bacteria-treated cysts showed delayed germination (10% germination rate) compared to control (above 65% germination rate). Following the recorded intervals at 36hr, and 48hr, the germination rate of treated groups was lower than 30%, and 40% respectively, while the control group showed more than 80% germination rate in both intervals. Major abnormalities were recorded separately including missing antennules, abnormal eyes, absent swimming legs, and a damaged ovary under a LABOMED CXL microscope (USA), as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2. Exponential Phase\u003c/h2\u003e\n \u003cp\u003eThe toxic effects of two coliform isolates were examined on \u003cem\u003eArtemia salina\u003c/em\u003e after germination. Treatments were applied in triplicate for each bacterium, along with controlled maintenance. All of the isolates exhibited the highest level of survival after an hour, and no significant variation was observed between the treatments and the control group (Figure. 4). \u003cem\u003eAcinetobacter\u003c/em\u003e sp. exhibited lower survival after 2 hours compared to \u003cem\u003eKlebsiella\u003c/em\u003e sp., although the difference between the two isolates was not statistically significant when compared to the control (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). We observed that, between 3 to 7 hours, the survival rate of \u003cem\u003eArtemia\u003c/em\u003e decreased over time, resulting in a variation that differed significantly from the control (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). After 8 hours, no \u003cem\u003eArtemia\u003c/em\u003e survived in the presence of \u003cem\u003eAcinetobacter sp.\u003c/em\u003e, and the variation was statistically significant (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Significant variations were also noticed using a LABOMED CXL microscope (USA), as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Determination of tD\u003csub\u003e50\u003c/sub\u003e\u003c/h2\u003e\n \u003cp\u003eThe tD\u003csub\u003e50\u003c/sub\u003e value for both \u003cem\u003eAcinetobacter\u003c/em\u003e sp. and \u003cem\u003eKlebsiella\u003c/em\u003e sp. was recorded during the exponential phase, which lasted 5 hours for \u003cem\u003eAcinetobacter\u003c/em\u003e sp. and 6 hours for \u003cem\u003eKlebsiella\u003c/em\u003e sp.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Determination of LC\u003csub\u003e50\u003c/sub\u003e\u003c/h2\u003e\n \u003cp\u003eThe cytotoxic effect of isolated bacteria was examined at different concentrations of 25, 50, 75, 100, 125, and 150 \u0026micro;l in this current investigation. The LC\u003csub\u003e50\u003c/sub\u003e value for \u003cem\u003eAcinetobacter\u003c/em\u003e sp., determined through the brine shrimp lethality bioassay, was 49.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u0026micro;l, with the regression equation Y\u0026thinsp;=\u0026thinsp;2.028x\u0026thinsp;\u0026plusmn;\u0026thinsp;1.565. The 95% confidence interval was between 31.11 and 78.49 \u0026micro;l after 24 hours of exposure (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Figure. S8). In contrast, the value for \u003cem\u003eKlebsiella\u003c/em\u003e sp. was 69.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u0026micro;l/ml, with a regression equation of Y\u0026thinsp;=\u0026thinsp;2.390x\u0026thinsp;\u0026plusmn;\u0026thinsp;0.589. The 95% confidence limits ranged from 50.11 to 97.79 \u0026micro;l/ml after 24 hours of exposure (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Figure S10). Additionally, the mortality rates of \u003cem\u003eArtemia salina\u003c/em\u003e induced by the isolated bacteria are presented in Figures S9 and S11, respectively.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eAfter a 24-hour exposure, the LC50 values, 95% confidence limits, regression equations, and Chi-square values for the isolated bacteria tested against brine shrimp nauplii are as follows:\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"112%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTest sample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e(\u0026mu;l)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% Confidence limits (\u0026mu;l)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRegression equation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026chi;\u003csup\u003e2\u003c/sup\u003e value (Degrees of freedom)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter\u0026nbsp;\u003c/em\u003esp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e61.07\u0026plusmn;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e43.73 to 85.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eY =2.532x\u0026plusmn;0.477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e0.585 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e69.99\u0026plusmn;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e50.11 to 97.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eY =2.390x\u0026plusmn;0.589\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. Pearson Correlation Between Dosses and Abnormality of Artemia salina\u003c/h2\u003e\n \u003cp\u003eUsing Pearson correlation, anomalies of treated \u003cem\u003eArtemia\u003c/em\u003e after 48 hours were found to be negatively correlated with controls in the case-stationary phase (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Similar significant correlations were also detected during the exponential phase (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). Moreover, the ANOSIM and ordination MDS identified the significant changes and separation between controls and treated \u003cem\u003eArtemia\u0026rsquo;s\u003c/em\u003e abnormalities (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb; Table S3 for ANOSIM).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7. Antibiotic Sensitivity Test\u003c/h2\u003e\n \u003cp\u003eThe sensitivity and resistance patterns of the isolated \u003cem\u003eAcinetobacter\u003c/em\u003e sp. confirmed that the isolated bacterium was resistant to ciprofloxacin, doxycycline, tetracycline, cefixime, ceftazidime, and penicillin (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) but susceptible to cefuroxime, kanamycin, gentamycin, amoxicillin, and erythromycin. On the contrary, \u003cem\u003eKlebsiella\u003c/em\u003e sp. was resistant to penicillin, tetracycline, doxycycline, and cefixime while susceptible to amoxicillin, kanamycin, ceftazidime, gentamycin, ciprofloxacin and cefuroxime (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"\" class=\"colspec\"\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eAntibiotic resistance profile of isolated bacteria.\u003c/div\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"105%\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName of \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Antibiotic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 32px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZone of inhibition (mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResistant pattern\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter\u0026nbsp;\u003c/em\u003esp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003ePenicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e8 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e9 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eAmoxicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e19 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e17 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eErythromycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e22 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e14 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eIntermediate resistant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eAmpicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e11 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e15 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eIntermediate resistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eIntermediate resistant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eKanamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e16 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e20 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eCeftazidime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e10 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e18 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eGentamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e17 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e22 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eTetracycline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e9 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e10 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eDoxycycline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e7 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e8 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eCiprofloxacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e8 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e23 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eCefuroxime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e16 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e17 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eCefixime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e6 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e9 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e Resistant=\u0026lt;10 mm; Intermediate =10-15 mm; Susceptible=\u0026gt;15 mm\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eFruit juices are a popular source of nutrients among people in developing countries like Bangladesh. The reason behind this is that fruit juices are delicious, accessible at low prices, and convenient for the consumers (Ohiokpehai \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Notably, the detection of coliform in fruit juice is not only permissible according to safe food consumption standards (Andres et al. 2004) [33], and regulations but also an indicator of other pathogens being present. Therefore, coliform is a significant marker as it helps raise awareness and determine the source of other pathogenic bacteria. Based on this approach, we isolated coliform bacteria from twelve fruit juice samples (local and packed) available in Rajshahi City. Among these samples, a lactose non-fermenting white-colored coliform colony (Isolate A) from sugarcane juice (Sample 6) and lactose fermenting pink-coloured colony (Isolate B) from packed mango drinks (Sample 9) were chosen for further research based on colony number and morphology.\u003c/p\u003e \u003cp\u003eMorphological features are key to determining the general characteristics of bacteria. The first report by Kabler and Clark (Kabler and Clark \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1960\u003c/span\u003e), showed that coliforms were rod-shaped, gram-negative, non-spore-forming bacteria that ferment lactose within 24 hours at a temperature of 37\u0026deg;C. Our findings also proved that the isolated bacteria were gram-negative, non-motile, and rod-shaped. Maximal growth was observed at different pH levels and temperatures. In this instance, isolate A exhibited the highest growth at a pH of 7.0 and a temperature of 35\u0026deg;C, whereas isolate B displayed maximum growth at a pH of 7.2 and a temperature of 37\u0026deg;C. According to Bergey\u0026rsquo;s Manual of Systematic Bacteriology (Garrity, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), the growth temperature and pH range for \u003cem\u003eAcinetobacter\u003c/em\u003e sp. is 35\u0026deg;-37\u0026deg;C and 7.0-7.5, respectively. Additionally, Bergey's Manual of Determinative Bacteriology (Breed et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1957\u003c/span\u003e) indicates that the pH range for \u003cem\u003eKlebsiella\u003c/em\u003e sp. is between 7.0 and 7.8, while the growth temperature range is from 30\u0026deg; to 37\u0026deg;C. Therefore, it is confirmed that the physiological and characteristic outputs of the isolated bacteria had a resemble the previous findings. Biochemical tests are among the principal methods for microbial identification (Atlas of Oral Microbiology 2015). Bergey\u0026rsquo;s Manual of Systemic Bacteriology (Garrity, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), confirmed the morphological and biochemical test results of the isolates as members of the genus \u003cem\u003eAcinetobacter\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e, respectively. The 16S rRNA gene sequencing is regarded as a favored genetic method for identification at the molecular level (Clarridge et al. 2004). Molecular identification indicated that bacterial isolate A had a 97% significant alignment with \u003cem\u003eAcinetobacter\u003c/em\u003e sp. (Accession no. LC437022), whereas isolate B had a 98% significant alignment with \u003cem\u003eKlebsiella\u003c/em\u003e sp. (Accession no. LC552682). Asif et al. reported \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e as a persistent infectious agent (Asif et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), while Martin and Bachman classified \u003cem\u003eKlebsiella pneumoniae as\u003c/em\u003e an opportunistic, hypervirulent, and multidrug-resistant pathogen based on a genome study (Martin and Bachman, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNeves et al. assessed acute dinoflagellate toxicity at a concentration of 200 cells ml⁻\u0026sup1;, which affected the germination and survival rates of \u003cem\u003eArtemia salina\u003c/em\u003e during the stationary and exponential phases, respectively (Neves et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). They also observed the highest tD\u003csub\u003e50\u003c/sub\u003e value for \u003cem\u003eG\u003c/em\u003e. \u003cem\u003eexcentricus\u003c/em\u003e within 4 hours in the case of the exponential phase. In our present study, we evaluated the toxicity of isolated bacteria, and developmental abnormalities were also identified in both the stationary and exponential phases of \u003cem\u003eArtemia salina\u003c/em\u003e at 200 cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Arulvasu et al. reported the impact of silver nanoparticles on both alive animals and cysts. In their findings, brine shrimp guts filled with nanoparticles showed a significant mortality level within 24 hours of exposure (Arulvasu et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePinheiro et al. reported the accumulation of MTiO\u003csub\u003e2\u003c/sub\u003e in \u003cem\u003eArtemia\u003c/em\u003e sp. after 24 hours and 48 hours without any morphological damage observed in the control group (Pinheiro et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In their experiment, MTiO\u003csub\u003e2\u003c/sub\u003e was found in the gut of \u003cem\u003eArtemia\u003c/em\u003e sp., and the cephalothorax showed swelling at concentrations of 50 ppm and 100 ppm, respectively. Additionally, the positive control K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e exhibited underdeveloped body growth within 24 hours of exposure. However, MTiO\u003csub\u003e2\u003c/sub\u003e dispersed throughout the body of \u003cem\u003eArtemia\u003c/em\u003e sp. resulting in morphological changes such as tissue degradation at the highest concentration of 100 ppm, while individuals exposed to K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e showed improper development with deformities in the appendages and abdomen. In the present study, during the stationary phase, isolated bacteria had a toxic effect on germination of \u003cem\u003eArtemia salina\u003c/em\u003e as both isolates showed delayed germination after 24 hours, while the control group showed germination after 16 hours. The variation between the treatment and control group was significant. In the case of the stationary phase, damaged germinated nauplii were found after 24 hours of \u003cem\u003eAcinetobacter\u003c/em\u003e sp. treatment, exhibiting missing antenulla, abnormal eyes, contused mandibles, absent swimming legs, and disrupted structure. After 48 hours of treatment with Acinetobacter sp. missing antenulla, absent swimming legs, and damaged ovaries were observed. Similarly, germinated deformed nauplii were observed after 24 hours of treatment with \u003cem\u003eKlebsiella\u003c/em\u003e sp., while missing antenulla, absent swimming legs, and damaged ovaries were observed after 48 hours. It was reported that \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e caused mortality in Nile tilapia (Silva et al. 2021). Bi et al. described disease-causing \u003cem\u003eAcinetobacter johnsonii\u003c/em\u003e in Rainbow Trout (Bi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAgain, during the exponential phase, the treatment with Acinetobacter sp. exhibited the highest level of toxicity. After 8 hours, it showed no survival with significant variation compared to the control group. Developmental abnormalities such as abnormal eye, improperly developed mandibles, absent swimming legs, defective structure, and abnormal telsion width were also identified after 8 hours of treatment. Moreover, \u003cem\u003eArtemia\u003c/em\u003e treated with \u003cem\u003eKlebsiella\u003c/em\u003e sp. exhibited low survival after 8 hours compared to previous hour, and the variation was significant. Developmental abnormalities missing antennula, deformed swimming legs, incorporeal ovary, and abnormal telsion width were also observed. In addition, the tD\u003csub\u003e50\u003c/sub\u003e was found to be within 5 hours for \u003cem\u003eAcinetobacter\u003c/em\u003e sp. and 6 hours for \u003cem\u003eKlebsiella\u003c/em\u003e sp., in the exponential phase. Similar abnormalities were found in a previous study by Talukder et al (Talukder et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and our findings aligned with their data. Therefore, it is evident that the toxicity level of \u003cem\u003eAcinetobacter\u003c/em\u003e sp. was higher than that of \u003cem\u003eKlebsiella\u003c/em\u003e sp. in both phases of \u003cem\u003eArtemia salina\u003c/em\u003e. Overall, the toxicity of isolated coliforms at the developmental level was significant and analyzed through DMRT. Furthermore, the ANOSIM result confirmed significant changes compared to the control group in both the stationary (R\u0026thinsp;=\u0026thinsp;0.646; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) phase and exponential phase (R\u0026thinsp;=\u0026thinsp;0.52; P\u0026thinsp;\u0026lt;\u0026thinsp;0.002). Moreover, the Pearson correlation validated the negative correlation between the control group and the treated \u003cem\u003eArtemia\u003c/em\u003e in both cases.\u003c/p\u003e \u003cp\u003eBrine shrimp bioassay is also used for cytotoxicity tests of microbial toxins and bioactive compounds. We also tested the toxicity of the isolates against \u003cem\u003eArtemia salina\u003c/em\u003e and the LC\u003csub\u003e50\u003c/sub\u003e values for \u003cem\u003eAcinetobacter\u003c/em\u003e sp. and \u003cem\u003eKlebsiella\u003c/em\u003e sp. were 61.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 \u0026micro;l and 69.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u0026micro;l, respectively after 24 hours of exposure. From the results, it was clear that \u003cem\u003eAcinetobacter\u003c/em\u003e sp. exhibited higher toxicity than \u003cem\u003eKlebsiella\u003c/em\u003e sp. against aquatic organisms.\u003c/p\u003e \u003cp\u003eWe identified several multidrug-resistant (MDR) isolates in the current investigation. Our results showed that \u003cem\u003eAcinetobacter\u003c/em\u003e sp. (Isolate A) was resistant to tetracycline, ciprofloxacin, penicillin, ceftazidime, cefixime, and doxycycline, while susceptible to kanamycin, gentamycin, erythromycin, amoxicillin, and cefuroxime, and had intermediate resistance only against ampicillin. In hospitalized patients, Dortet et al. isolated \u003cem\u003eAcinetobacter ursingii\u003c/em\u003e and \u003cem\u003eAcinetobacter schindleri\u003c/em\u003e that were resistant to cefixime and susceptible to amoxicillin, kanamycin, and gentamycin (Dortet et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The previous report found multidrug-resistant \u003cem\u003eAcinetobacter\u003c/em\u003e sp. from fruits, which exhibited 60% resistance against ceftazidime and 84% resistance against ciprofloxacin (Carvalheira, 2017). Another investigation carried out by Pal et al. reported that \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e was 100% resistant against penicillin (Pal et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), followed by Ferdous et al. who showed a resistance pattern of \u003cem\u003eAcinetobacter\u003c/em\u003e sp. isolated from the hospital exhibiting 68.5% resistance against tetracycline, 92% resistance against ampicillin, and 84.9% resistance against ciprofloxacin (Ferdous et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). So, it is confirmed that our results are in accordance with previous findings. On the other hand, our results depicted that \u003cem\u003eKlebsiella\u003c/em\u003e sp. (Isolate B) was resistant against penicillin, tetracycline, doxycycline, and cefixime, intermediately resistant against ampicillin, and erythromycin, but susceptible to amoxicillin, kanamycin, ceftazidime, gentamycin, ciprofloxacin, and cefuroxime. According to Khalif et al. an antibiogram study of pathogenic bacteria from street-vented food showed that \u003cem\u003eKlebsiella\u003c/em\u003e sp. was susceptible to kanamycin, gentamycin, and ciprofloxacin, but resistant to erythromycin (Khalif et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Another study revealed that multidrug-resistant \u003cem\u003eKlebsiella pneumonae\u003c/em\u003e isolated from powdered infant formula showed 15.4% resistance against cefixime, 53.8% resistance against ampicillin, 38.5% susceptibility to cefuroxime, 84.6% susceptibility to ceftazidime, and 100% susceptibility to ciprofloxacin, gentamycin, and kanamycin (Zhou et. al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In addition, Padamadan et al. (Padamadan et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported an antibiogram of street-vended and packed fruit juices in which \u003cem\u003eKlebsiella\u003c/em\u003e sp. was resistant against ampicillin, cefixime, and penicillin. Hence, it can be concluded that our research output had similarities with the referred data. From the results, it is clear that \u003cem\u003eAcinetobacter\u003c/em\u003e sp. was more resistant to antibiotics in contrast to \u003cem\u003eKlebsiella\u003c/em\u003e sp. So, both screened bacteria might be a matter of health issues without proper consciousness as well as without assuring the hygienic status of juice during processing, storage, selling, and drinking by children or people.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eFrom the results of the current study, it has been confirmed that fruit juices (both street-vented and packaged) show higher levels of contamination with pathogens, including \u003cem\u003eAcinetobacter\u003c/em\u003e sp. and \u003cem\u003eKlebsiella\u003c/em\u003e sp. Additionally, these indicative pathogens have shown resistance to multiple antibiotics. The DMRT analysis revealed notable abnormalities in \u003cem\u003eArtemia salina\u003c/em\u003e due to multidrug-resistant coliforms in terms of coliform developmental toxicity. Furthermore, ANOSIM and ordination MDS indicated significant abnormalities among bacteria-treated \u003cem\u003eArtemia\u003c/em\u003e compared to the control group. Pearson correlation analysis showed that, longer exposure times of coliforms to \u003cem\u003eArtemia salina\u003c/em\u003e were significantly positively correlated with abnormalities when compared to the control group. Since fruit juices are popular beverage, their quality is of utmost importance to human health. The microbial contamination of these juices poses a serious threat as it continues to be a significant source of illness, particularly among children, and affect the health of population in both developed and developing regions. Therefore, storage strategies should be implemented to ensure the microbial standards of street-vented and commercially available fruit juices, in order to guarantee food security and safety. This way, the spread of antibiotic-resistant infections within the population can be prevented.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eEthical approval was not applicable to this research as it did not involve human participants, animals, or related ethical concerns.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e All data can be obtained from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatents:\u003c/strong\u003e Not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work received no external funding from any sources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Consent to Participate:\u003c/strong\u003e This study did not involve human participants; therefore, a \u0026apos;Consent to Participate\u0026apos; declaration is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, M.N.H., and M.A.E.; Methodology, M.N.H., M.A.E., and A.P.T.; Data analysis, M.A.E., and M.N.H.; Data curation, M.N.H., M.A.E., A.P.T., and S.S.D.; Writing-original draft, M.N.H., A.P.T., and S.S.D.; Writing-reviewing and editing, M.A. S., M.A. E.; Visualization, M.A.S. and S.Z.; Supervision, M.S.U., and M.A.E.; Laboratory facilities, M.A.E., M.A.S., M.S.U., and S.Z. All of the authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eC\u003cstrong\u003eonflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbbo, M.; Olurin, T.O.; Odeyemi, G. Studies on the storage stability of soursop (\u003cem\u003eAnnona muricata\u003c/em\u003e L.) juice. \u003cem\u003eAfr. J. Biotechnol.\u003c/em\u003e \u003cstrong\u003e2006\u003c/strong\u003e, \u003cem\u003e5\u003c/em\u003e, 108-112.\u003c/li\u003e\n \u003cli\u003eAnderson, J.E.; Goetz, C.M.; McLaughlin, J.L.; Suffness, M. A blind comparison of simple bench-top bioassays and human tumour cell cytotoxicities as antitumor prescreens. \u003cem\u003ePhytochem. Anal\u003c/em\u003e. \u003cstrong\u003e1991\u003c/strong\u003e, \u003cem\u003e2\u003c/em\u003e, 107-111.\u003c/li\u003e\n \u003cli\u003eAndr\u0026eacute;s, S.C.; Giannuzzi, L.; Zaritzky, N.E. 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The developmental toxicity, bioaccumulation and distribution of oxidized single walled carbon nanotubes in \u003cem\u003eArtemia salina\u003c/em\u003e. \u003cem\u003eToxicol. Res.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e7\u003c/em\u003e, 897-906.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"fruit juice, multidrug-resistant (MDR) Artemia salina, deformities, pearson correlation","lastPublishedDoi":"10.21203/rs.3.rs-6737003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6737003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrobial spoilage of fruit juice is one of the major concerns for human infections in Bangladesh. In this context, local and packaged fruit juices were microbiologically assessed based on lactose-fermenting ability. The isolates were tested to analyze acute toxicity against the developmental stages of \u003cem\u003eArtemia salina\u003c/em\u003e and correlations between the bacterial dose duration and brine shrimp\u0026rsquo;s abnormalities. The morphological, physiological, biochemical, and 16S rRNA sequencing revealed that isolate A was \u003cem\u003eAcinetobacter\u003c/em\u003e sp. (lactose-non-fermenting, sample-6) and B was \u003cem\u003eKlebsiella\u003c/em\u003e sp. (lactose-fermenting, sample-9). The developmental toxicity of both bacteria induced delayed germination of \u003cem\u003eArtemia\u003c/em\u003e cysts and after 48 hours, missing antenulla, abnormal eye, contused mandibles, absent swimming legs, and damaged ovaries were recorded. The Duncan Multiple Range Test confirmed the highest toxicity of \u003cem\u003eAcinetobacter\u003c/em\u003e sp. compared to \u003cem\u003eKlebsiella\u003c/em\u003e sp. The time of death (tD\u003csub\u003e50\u003c/sub\u003e) was 5 hours for \u003cem\u003eAcinetobacter\u003c/em\u003e sp. and 6 hours for \u003cem\u003eKlebsiella\u003c/em\u003e sp. The lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e) values were 61.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u0026micro;l and 69.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u0026micro;l, respectively. Pearson correlations revealed defective traits were positively correlated with the increased coliform\u0026rsquo;s exposure duration to \u003cem\u003eArtemia salina\u003c/em\u003e and multidimensional test confirmed complete separation between control and treated \u003cem\u003eArtemia\u003c/em\u003e. Both bacteria were multidrug-resistant. This study suggests that awareness should be raised to eradicate microbial health hazards.\u003c/p\u003e","manuscriptTitle":"Screening of multidrug-resistant coliforms from fruit juice and their significant correlations with the developmental deformities of Artemia salina (Brine shrimp)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 08:05:54","doi":"10.21203/rs.3.rs-6737003/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6e421261-8a9a-46f9-8d77-191b16c23dd6","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T08:05:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 08:05:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6737003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6737003","identity":"rs-6737003","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}