Introduction
Water is vital to life, nevertheless many people do not have access to clean and safe drinking water and many die of waterborne bacterial infections. Several diseases can be evoked by pathogenic microorganisms found in contaminated water.1 In this context, water resources are the main stream carter of water borne pathogens which finally have adverse impact on human health and also on socio-economic environment. Most importantly, these pathogens are diverse in nature and placing entire communities at jeopardy.2 Water quality assessing depends on the identification of disease producing microorganisms present in water and the best approach is the use of an easily measured “indicator organism” to signal that pathogenic microorganisms may be present and the coliform group of bacteria is the marker used worldwide.3 Coliform bacteria include a large group of many types of bacteria (Coliform are defined as aerobic and facultative anaerobic bacteria, gram-negative, nonspore-forming and rod-shaped bacteria that ferment lactose with gas and acid formation within 48 hour when incubated at 37°C).4 Most coliforms are present in large numbers among the intestinal flora of humans and other warm blooded animals and are therefore found in fecal wastes5 and fecally contaminated drinking water is a main public health setback and cause of water transmit diseases.6
In developing countries, waterborne diseases are the major matter of concern regarding public health.7 Worldwide, the burden of diarrheal disease is highest in Southeast Asia and Africa8 and unsafe drinking water is the main cause of 1.8 million deaths of children aged below 5 years due to diarrheal diseases yearly.9 If coliform bacteria are present in drinking water, risk of contracting a waterborne illness is raised. The main bacterial diseases transmitted through drinking water include Cholera, Typhoid fever, Bacillary dysentery or Shigellosis, Acute diarrheas, Gastroenteritis and other serious Salmonellosis.10 Outbreaks of disease attributable to drinking water can escort to serious acute, chronic, or sometimes fatal health consequences.
In this perspective, Bangladesh is in a great peril as a developing country with uppermost density of population in the world. The percentage of utilizing ground water for drinking and household use is about 97% of the population of the country.11 Scarcity of safe drinking water is a common problem for both the urban and rural areas in Bangladesh.12 The mainstream (64%) of the urban population and almost all (93%) of the rural population have access to hand-pumped or piped water due to bacteriological contamination of surface water in Bangladesh.13 Several studies have confirmed that surface water sources in Bangladesh are profoundly contaminated with fecal coliforms (FC) and by various pathogenic bacteria.14 Most notably, environmental enteric dysfunction is an abnormality of gut function that might explain why most nutrition interventions fail to normalize early childhood growth.15 It is reported that improvements to drinking water quality, sanitation, and hand washing might develop the effectiveness of nutrition interventions and thereby help to deal with a larger portion of the observed growth deficit.16
In recent years, bottled water consumption has increased significantly in Bangladesh. Presence of fecal indicator and heterotrophic bacteria have been reported with levels exceeding drinking water guidelines by various investigations.17 Local newspapers and social media in Bangladesh have expressed their deep concerns that some brands of bottled water may be unsafe for consumption.18 Pathogenic bacteria such as Aeromonas sp.,19 Pseudomonas sp.,20 Shigella sp.,21 Salmonella sp.,22 Vibrio cholera23 etc have been perceived in bottled water. European Community Directive (European Community 1980) reported that total coliforms, E.coli, Enterococcus sp., Pseudomonas aeruginosa and parasites should not be identified in 250 ml of bottled water and World Health Organization24 suggests that the number of fecal coliforms should be zero in drinking water. Hence, the present study was planned on the isolation, characterization, profiling resistance pattern of coliform bacteria from drinking water which acts as an “indicator organism” of other pathogenic bacteria and assessing their toxicity on the developmental stages of aquatic organism Artemia salina and so on.
Materials and Methods
Sample collection
Total twelve drinking water samples were collected during March, 2017. Among twelve samples, four were local drinking water samples (Sample1- Sample 4) which were collected from different areas of Rajshahi University campus and eight were bottled water samples (Sample 5- Sample 12) which were collected from different regions of Rajshahi. Samples were aseptically collected and brought to the Microbiology Laboratory, Dept. of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi, Bangladesh for further investigation.
Isolation and optimization of coliform bacterial strains
Separately, each sample was used for plating on selective media. In this case, MacConkey agar media (Peptones 3.000 mg/L; Pancreatic digest of gelatin 17.000 mg/L; Lactose monohydrate 10.000 mg/L; Bile salts 1.500 mg/L; Sodium chloride 5.000 mg/L; Crystal violet 0.001 mg/L; Neutral red 0.030 mg/L; Agar 13.500 mg/L) was used. Plates were incubated at 37°C for 24 hour. Then, coliform bacteria were identified on the basis of colony morphology and lactose fermenting ability. Finally, isolates were maintained as pure cultured strains and preserved for further experiments. The effects of pH and temperatures on growth of isolated bacteria were optimized at 660 nm wavelength using spectrophotometer (Analytik Jena, Germany).
Morphological and biochemical characterization of isolated bacteria
Morphological and biochemical tests were done for the specific identification and characterization of bacteria. Isolated bacteria were characterized by several morphological (Gram staining and motility) and biochemical (Methyl red, catalase, starch agar, mannitol salt agar, TSI, simmons citrate agar and urea agar) tests.
Molecular identification of isolated bacteria
Molecular identification and characterization of the isolates were performed through the following steps: extraction of chromosomal DNA,25 amplification of 16S rRNA gene sequence, purification of PCR products, cycle sequencing, purification of cycle sequencing products, detection of nucleotides and sequence analysis. 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used as forward and reverse primer, respectively. 16S rRNA gene sequences of selected bacterial isolates were compared with other reference sequences as available in the NCBI database using the Basic Local Alignment Search Tool (BLAST) algorithm.
Antibiotic sensitivity test and determination of minimum inhibitory concentration (MIC)
Antibiotic sensitivity and resistance of the isolated bacteria were assayed according to the Kirby- Bauer disc diffusion method.26 It was done against penicillin, amoxicillin, erythromycin, ampicillin, kanamycin, ceftazidimde, doxycycline, gentamycin, tetracycline, ciprofloxacin, cefuroxime and cefixime, respectively. Zones of inhibition were measured with the help of mm scale.
Minimum Inhibitory Concentration (MIC) of isolated bacteria was carried out through tube dilution method.27
Assessing toxicity of isolated bacteria on the developmental stages of Artemia salina
Artemia salina is commonly used for toxicity tests owing to its extensive distribution, short life cycle, non-selective grazing and sensitivity to toxic substances.28 Acute toxicity assays were performed with Artemia cysts in two phases such as stationary (up to 48 hour) and exponential (up to 8 hour) to assess differences in cell toxicity of coliform bacterial strains isolated from drinking water.
Stationary phase
The stationary phase of Artemia cysts was maintained up to 48 hour at appropriate cyst germination laboratory condition (25°C and aeration pump) to assure well germination with 3 replications and control. Germination of cysts with each bacteria (O.D. = 0.5) and controls with triplicate were observed under inverted microscope (LABOMED CXL, USA) after every six hours of incubation. Data were kept and photographs of abnormalities were taken under a microscope (LABOMED CXL, USA).
Exponential phase
The exponential phase of Artemia salina was maintained up to 8 hour at appropriate laboratory condition (25°C and aeration pump) with 3 replications and control. Intoxication assays were performed in 6-well plates where each well contains 10 ml of filtered seawater, six Artemia salina. Then, the well plates were incubated for 8 hour with overnight cultured isolated bacteria (O.D.= 0.5) in abundances of 200 cells ml-1. Each treatment containing the brine shrimps and one isolated bacteria was performed in triplicate and the control without the isolated bacteria was also maintained in triplicate. Abnormalities and survival of individuals were verified under inverted microscope (LABOMED CXL, USA) after every hour of incubation.
Determination of tD50
The tD50 value was also determined in exponential phase. It is the time (hour) when 50% Artemia salina died in case of exponential phase.
Statistical analysis
Statistical analysis was done using Statistical Analysis Software (SAS), version 9.1.3 by Duncan Multiple Range Test (DMRT).
Determination of LC50
Toxicity of three isolated bacteria were evaluated through detection of LC50 at concentrations of 25 µl, 50 µl, 75 µl, 100 µl, 125 µl, 150 µl. At first, simulated sea water was taken in a small tank and shrimp eggs (1.5 gm/l) were added to one side of the perforated divided tank with constant oxygen supply to get nauplii. Finally, 10 ml of simulated sea water solution with 10 nauplii was added to each of the test tube. The test tubes were left uncovered under the lamp and an incubation period of 24 hours was given at room temperature for observation. For each concentration, one vial containing 10 ml sea water and 10 shrimp nauplii were used as positive control group. It was used to verify the validity of the test. After 24 hours, the vials were observed. The number of survived nauplii in each vial was counted and the results were noted. The probit analysis was carried out through the Finney method to determine LC50.29
Results
Isolation of coliform bacteria and optimization at different pH and temperature
Among twelve samples, highest number of coliform colonies were found from Sample 1 (local drinking water) then Sample 7 (bottled water), Sample 9 (bottled water) and were selected for further investigation on the basis of colony number and morphology but Sample 5 (bottled water) showed no growth on MacConkey agar media (Figure 1). Isolate A (Aeromonas sp.) showed highest growth at pH 7.2 and 35°C (Figure 2,Figure 3); Isolate B (Enterobacter sp.) showed maximum growth at pH 7.0 and 30°C (Figure 4,Figure 5) while Isolate C (Escherichia sp.) revealed utmost growth at pH 7.4 and 35°C (Figure 6,Figure 7).
Figure 2
Effect of optimum pH level on the growth of Isolate A (Aeromonas sp.) upto 72 hours of incubation period
Figure 3
Effect of temperature variations on the growth of Isolate A (Aeromonas sp.) upto 72 hours of incubation period
Figure 4
Effect of optimum pH level on the growth of Isolate B (Enterobacter sp.) upto 72 hours of incubation period
Figure 5
Effect of temperature variations on the growth of Isolate B (Enterobacter sp.) upto 72 hours of incubation period
Figure 6
Effect of optimum pH level on the growth of Isolate C (Escherichia sp.) upto 72 hours of incubation period
Figure 7
Effect of temperature variations on the growth of Isolate C (Escherichia sp.) upto 72 hours of incubation period
Morphological and biochemical characterization of isolated bacteria
Morphological characteristics indicated that all three isolates were motile, gram negative, rod shaped bacteria. Biochemical tests confirmed that isolate A was lactose fermenting, gas non-producing; methyl red, catalase and starch hydrolysis test positive; simmmons citrate, urea hydrolysis and mannitol salt agar test negative. Isolate B was lactose non- fermenting, gas producing; catalase, simmons citrate, urea hydrolysis and starch hydrolysis test positive; methyl red and mannitol salt agar test negative while isolate C showed positive result in all tests.
Molecular identification of isolated bacteria
When the 16S rRNA gene sequences of isolated bacteria were verified with the 16S rRNA gene sequences of other organisms that had already been submitted to NCBI Gene bank database using the BLASTN (http://www.ncbi.nih.gov/BLAST) algorithm, it indicated that isolate A showed 76% identity with Aeromonas sp. (Accession no. LC435695), isolate B showed 96% identity with Enterobacter sp. (Accession no. LC434449) while isolate C showed 96% identity with Escherichia sp. (Accession no. LC384629). DNA quantification analysis and PCR band of isolated bacteria were shown in Figure 8,Figure 9.
Antibiotic sensitivity test and determination of minimum inhibitory concentration
The result showed that, Aeromonas sp. and Escherichia sp. were multi drug resistant. Aeromonas sp. was resistant against penicillin, ceftazidime, doxycycline, cefuroxime and cefixime (Table 1). However, Escherichia sp. was resistant against penicillin, ceftazidime, doxycycline and cefuroxime (Table 1). On the other hand, Enterobacter sp. was resistant against ceftazidime and cefuroxime (Table 1).
Figure 14
Regression line of log dose and probit mortality of Aeromonas sp. against brine-shrimp nauplii after 24 hours of exposure
Stationary phase
After 16 hour, all three isolates, Aeromonas sp., Enterobacter sp. and Escherichia sp. treated Artemia cyst showed no germination (Figure 10). After 24 and 36 hour, Aeromonas sp. and Enterobacter sp. treated cyst showed germination but Escherichia sp. treated cyst showed no germination and the variation was significant between the treatments compared to control (Figure 10). However, after 48 hour, all the isolates showed germination and the variation was significant among control with all three bacteria treated cyst (Figure 10). Major abnormalities were observed under microscope (LABOMED CXL, USA) which was shown in Figure 11.
Table 1
Antibiotic sensitivity test used for the detection of the resistance pattern of the isolated bacteria
The minimum inhibitory concentrations of Aeromonas sp., Enterobacter sp. and Escherichia sp. against gentamycin were 6.25 μg/ml, 12.5 μg/ml and 6.25 μg/ml, respectively (Table 2).
Table 2
The minimum inhibitory concentration of isolated bacteria against gentamycin
The minimum inhibitory concentrations of Aeromonas sp., Enterobacter sp. and Escherichia sp. against amoxicillin were observed 1.56 μg/ml, 12.5 μg/ml and 3.125 μg/ml, respectively (Table 3).
Table 3
The minimum inhibitory concentrations of isolated bacteria against amoxicillin
Table 4
| Growth Phase | (Aeromonas sp.) | (Enterobacter sp.) | (Escherichia sp.) |
| Exponential | 6 | 7 | 4 |
Time (hr) when 50% Artemia salina died (tD50) in exponential phase
Table 5
LC50, 95% confidence limits, regression equations and Chi-square values for isolated bacteria against brine shrimp nauplii after 24 hours of exposure
Exponential phase
Toxic effects of the three coliform isolates were conducted on germinated Artemia salina and treatment was added with three replications for each bacterium as well as control maintained. After 1 hour, all isolated bacteria treated Artemia showed highest level of survival and the variation was non-significant among the treatments in comparison to control (Figure 12). After 2 hour, bacteria treated Artemia showed low survival than 1 hour and the variation was significant among treatments compared to control (Figure 12). Again, after 3-7 hour, we found that the survival of Artemia was decreased with time and the variation was risen significantly in comparison to control (Figure 12). However, after 8 hour, no survival was found in case of treatment with Escherichia sp. and the variation was significant. Major abnormalities were also observed under microscope (LABOMED CXL, USA) which was shown in Figure 13.
Determination of tD50
tD50 value was observed in case of exponential phase which were 6 hour, 7 hour and 4 hour for Aeromonas sp., Enterobacter sp. and Escherichia sp., respectively (Table 4).
Determination of LC50
Isolated Aeromonas sp., Enterobacter sp. and Escherichia sp. showed 50% toxicity against aquatic organism at the concentration of 56.26±0.18, 85.41±0.23 and 48.32±0.17 μl, respectively (Table 5). Regression line of log dose and probit mortality of isolated bacteria against brine- shrimp nauplii was shown in Figure 14,Figure 15,Figure 16.
Discussion
Several studies have reported isolation and characterization of coliform bacteria from drinking water. Parvez et al. outlined bacteriological quality of drinking water samples a cross Bangladesh.30 Ahmed et al. also published article on the isolated fecal indicators and bacterial pathogens in bottled water from Dhaka, Bangladesh.31 However, our study was designed not only to coliform bacterial strains (Isolate A as Aeromonas sp., Isolate B as Enterobacter sp. and Isolate C as Escherichia sp.) isolation, physiological study and profiling resistance pattern but also for the evaluation of their acute toxicity on the developmental stages of aquatic organism especially on Artemia salina. Morphological identification showed all three isolates were motile, gram negative and rod shaped bacteria. Biochemical characteristics indicated that isolate A was lactose fermenting, gas non-producing, methyl red, catalase and starch hydrolysis test positive; isolate B was lactose non- fermenting, gas producing, catalase, simmons citrate, urea hydrolysis and starch hydrolysis test positive while isolate C confirmed positive result for all tests. In our investigation, all three isolates illustrated comparative differences in the growth performances. They showed maximal growth at different pH and temperature. However, Isolate A revealed maximum growth at pH 7.2 and in 35°C, Isolate B at pH 7.0 and in 30°C while Isolate C at pH 7.4 and in 35°C. Following Bergey’s Manual of Systematic Bacteriology32 (Second Edition), isolate A showed similar characteristics of the genus Aeromonas, isolate B showed similar features of the genus Enterobacter while isolate C revealed similarity of the genus Escherichia. So, it can be assumed that outputs of physiological and biochemical features of the isolates have similarity with the referred data.
Comparison of the bacterial 16S rRNA gene sequencing has materialized as a preferred genetic technique in molecular biology.33 Thus, in the present research, sequencings were done by 16S rRNA gene for molecular identification of the isolated bacterial strains. Sequencing result of 16S rRNA gene using the Basic Local Alignment Search Tool (BLAST) algorithm authenticated that the bacterial isolate A showed 76% significant alignments with Aeromonas sp., isolate B showed 96% significant alignments with Enterobacter sp. strain and isolate C showed 96% significant alignments with Escherichia sp. Merih et al. monitored the occurrence of pathogenic Aeromonas sp. in publi c drinking water.34 Arun et al. also investigated the exsistence of enteric pathogens such as Escherichia coli and Enterobacter aerogenes from different drinking water sources.35 Our sequencing results showed resemblance with the previously isolated coliforms.
Availability of multiple drug resistance (MDR) coliform was reported in tubewell water.36 They found that all of the coliform isolates were resistant to ampicillin, all fecal coliform isolates to penicillin and sulphamethoxazole. Another study of quality analysis of Dhaka WASA drinking water by Mahbub et al. showed that all eight E. coli isolates were found resistant to ampicillin, amoxicillin, kanamycin, penicillin antibiotics and almost all of them were found sensitive to gentamycin, nalidixic acid and ciprofloxacin.37 Beside these, Scoaris et al.reported multiple drug resistance Aeromonas sp.38 and prevalence of multidrug resistant Escherichia coli from drinking water sources were also investigated.39 Antibiotic resistant Enterobacter sp. was also found from raw source water and treated drinking water.40 In the present study, multiple drug resistance (MDR) isolates were found. Among three isolates, Aeromonas sp. and Escherichia sp. exhibited resistance to multiple antibiotics while Enterobacter sp. was resistant against only two antibiotics. Aeromonas sp. was resistant to penicillin, ceftazidime, doxycycline, cefuroxime, cefixime and Escherichia sp. was resistant to penicillin, ceftazidime, doxycycline and cefuroxime but Enterobacter sp. was resistant to only ceftazidime and cefuroxime. Thus, the resistance was showed as Aeromonas sp.> Escherichia sp.> Enterobacter sp. in this pattern. All the isolates were susceptible to amoxicillin, erythromycin, kanamycin, gentamycin, tetracycline and ciprofloxacin. Moreover, the MIC value of gentamycin against Aeromonas sp., Enterobacter sp. and Escherichia sp. were 6.25 μg/ml, 12.5 μg/ml and 6.25 μg/ml, respectively. The MIC value of another antibiotic amoxicillin was also determined and the MIC concentrations for the Aeromonas sp., Enterobacter sp. and Escherichia sp. were found as 1.56 μg/ml, 12.5 μg/ml and 3.125 μg/ml, respectively. From the test, it was confirmed that between these two antibiotics, Aeromonas sp. and Escherichia sp. can be easily controlled by amoxicillin (β-lactum class) in contrast to gentamycin (amino glycosides class). Our result confirmed promising outcome to control coliforms in future aspect.
Supratik Kar et al. proposed interspecies cytotoxicity parallel models between Escherichia coli (prokaryotic system) and human cell line (HaCaT) (eukaryotic system).41 Previously, Neves et al. reported acute toxicity of dinoflagellate cells in two growth phases of Artemia salina at 200 cells ml-1 to assess differences in cell toxicity to Artemia salina.42 In their study, the toxic benthic dinoflagellates significantly affected the mortality and survival rates of Artemia salina in stationary and exponential phase, respectively. Highest tD50 value was also found 4 hour for brine shrimps depicted to G. excentricus in exponential phase. Notably, we performed bacterial toxicity assays on Artemia salina in stationary and exponential phase with 200 cells ml-1 of isolated bacteria. No previous data about the toxic effect of coliform bacteria on the developmental stages of Artemia salina was reported before our investigation. In case of stationary phase, all bacterial treated cysts showed preferably delayed germination. In addition to that other abnormalities were detected among bacteria treated germinated cysts such as missing antennula, improper developed mandibles, abnormal telson width were photographically recorded when treated by Aeromonas sp. and fragile swimming legs, deformed ovary were captured by Enterobacter sp. treatment and abnormal eye, contused mandibles, damaged ovary were confined by Escherichia sp. treatment. Our results also illustrated significant level of developmental abnormalities at exponential phase of Artemia salina by the isolates in comparison to control. Deformed structure was observed in case of Aeromonas sp. treatment; deformed antennula, abnormal eye, damaged swimming legs were noticed in case of Enterobacter sp. treatment; abnormal head width, swimming leg missing, abnormal ovary were found in case of Escherichia sp. treatment. The tD50 was also experimented in exponential phase. In case of Aeromonas sp., tD50 was found within 6 hour, Enterobacter sp. within 7 hour while Escherichia sp. within 4 hour which showed similarity with referred data. Remarkably, variation in germination and survival of Artemia salina were increased with time among all three treatments in comparison to control and it was confirmed by DMRT analysis. From the result, it was clear that highest level of toxicity was observed in case of Escherichia sp. in both phases (stationary and exponential).
In our study, the LC50 results of the isolates were also evaluated. LC50 values of Aeromonas sp., Enterobacter sp. and Escherichia sp. were 56.26±0.18 m l, 85.41±0.23 µl and 48.32±0.17 µl after 24 hours, respectively. Harwig and Scott described 50% mortality (LC50 value) of known mycotoxins for 16 hour against Artemia salina which was 1.3 µg/ml for aflatoxin G1, 3.5 µg/ml for gliotoxin and 10.1 µg/ml for ochratoxin A.43 From our data, it was clear that the bacterial isolates had cytotoxic effect against the aquatic organism A. salina and the toxicity level was marked as Escherichia sp.> Aeromonas sp. > Enterobacter sp. after 24 hour of exposure on Artemia salina which showed less toxicity than the referred toxins data.
Conclusion
The antibiotic resistance patterns of these isolated gram-negative bacterial strains are correlated well, perhaps indicating their common origin and mode for emerging as antibiotic resistance strains. On the other hand, DMRT analysis indicated significant abnormalities in Artemia salina caused by multidrug coliforms and enunciate that isolated bacteria would have harmful effect for human health which can cause serious water borne diseases. Therefore, development and spread of antibiotic resistance in bacteria should pay attention while prescribing antibiotics to treat patients suffering from infection caused by pathogenic bacteria isolated from the studied samples.
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