The widespread emergence of antibiotic resistance among bacterial pathogens has become one of the most serious challenges in Ethiopia. This study determined the prevalence and drug resistance patterns of bacterial pathogens isolated from treated and untreated wastewater released from Ayder Referral Hospital in Northern Ethiopia. A cross sectional study design was conducted from September-December, 2015 in wastewater released from Ayder referral hospital. A total of 40 composite samples were aseptically collected, transported and processed for enumeration of indicator organisms, bacteriological identification and susceptibility testing following standard procedure. Data obtained were entered and analyzed using SPSS version 20. Mean heterotrophic plate count, total coliform count, fecal coliform count and E. coli count were found to be 1.6 × 106 CFU/mL, 2.2 × 106 CFU/100 mL, 2.0 × 105 CFU/100 mL and 1.1 × 104 CFU/100 mL from treated wastewater respectively. Among the total samples 134 bacterial isolates were detected and [84 (62.7%)] were from untreated wastewater and [50 (37.3%)] were from treated wastewater. The most frequently isolated bacteria from untreated wastewater samples was Klebsiella spp [14 (16.7%)] followed by S. aureus [13 (15.5%)] and P. aeruginosa [12 (14.3%)], similarly in treated wastewater samples Klebsiella spp [10 (20%)], P. aeruginosa [8 (16%)] and S. aureus [8 (16%)] were frequently detected. The overall multi-drug resistance (MDR) in this study was [79/134 (79.1%)]. MDR from untreated wastewater sample was [64/84 (76.2%)] while from treated wastewater sample was [42/50 (84%)] and shows significant difference with (COR: 1.64, 95% CI: 1.15 - 3.29, P: 0.001). It is concluded that treated hospital wastewater contains large numbers of antibiotic resistant bacteria. Therefore, there should be continuous monitoring and evaluation of the effluent quality of the ponds and chlorination of the final effluent should be developed.
Wastewater is an ideal media for a wide range of microorganisms especially bacteria, viruses and protozoa, and used as a reservoir for resistant bacteria [
The volume of antibiotics used in hospitals released into effluent results a selection pressure on bacteria. Therefore, wastewater from hospitals contains high numbers of resistant bacteria and antibiotic residues at concentrations able to inhibit the growth of susceptible bacteria [
Ayder referral hospital has wastewater stabilization pond as treatment system. The treatment plant consists of seven rectangular slanty ponds. The ponds are lined at the bottom by thick plastic to minimize seepage into ground water zones. The first pond (facultative pond) is the largest of all, having greater length and width from the other ponds and receives raw wastewater. It is primarily designed for BOD removal and has three zones in vertical section; those are supper aerobic zone, middle facultative zone or intermediate zone and lower anaerobic zone. Maturation ponds consist of six slanty ponds having almost similar length and width and have shallow depth. It receives wastewater effluent from facultative pond and its primary function is removal of pathogenic microorganisms. The last step in treatment process is release of treated effluent to open field continuously after flowing approximately 50 meter sewage system. The Untreated Hospital Wastewater (UHWW) sampling site was just the inlet of treatment plant while the Treated hospital Wastewater (THWW) sampling site was just at the outlet of treatment plant before released to the open field.
Cross sectional study design was conducted to collect hospital wastewater samples from September, 2015 to December, 2015.
A “Composite-sampling” technique was applied to collect the most representative samples according to guidelines of wastewater sampling techniques stated on APHA [
Total heterotrophic plate count: Duplicate plates were prepared for each volume of sample examined. All the samples were vigorously shaken before preparation of dilutions then serial 10-fold dilutions of samples were prepared in physiological saline, and 1 mL aliquot was spread over nutrient agar plate (Himedia). Then plates were incubated for 48 h at 37˚C before bacteriological counts were performed. Number of colonies on duplicate plate having 30 - 300 colonies was counted and finally bacterial count was reported as CFU/ mL.
Total coliform count: Serial 10-fold dilutions of sample were prepared in physiological saline and 1mL of aliquot was transferred aseptically in to a series of 9 test tubes containing Durham tubes and double strength MacConkey broth (Himedia). Tubes were gently shaken and incubated for 24 - 48 h at 37˚C, then production of gas and lactose fermentation was taken as positive reaction. Finally bacterial load were estimated using MPN table and dilution factor. The bacterial count was reported as CFU/100 mL.
Fecal coliform count: Serial 10-fold dilutions of sample were prepared in physiological saline and 1mL of aliquot was transferred aseptically in to a series of 9 test tubes containing Durham tubes and double strength MacConkey broth. Tubes were gently shaken and incubated for 24 h at 44.5˚C in water bath, then production of gas and lactose fermentation was taken as positive reaction. Furthermore E. coli cont was confirmed by adding approximately 0.1 ml of Kovacs reagent (Uni-Chem). Finally bacterial load were estimated using MPN table and dilution factor. The bacterial count was reported as CFU/100 mL.
Culture technique: The sample was investigated for further isolation and identification of bacterial pathogens. Representative diluted sample of 1 ml aliquots was plated on selective and differential media. MacConkey agar (SRL-sisco research laboratory), Salmonella-shigella agar (Uni-Chem) and Mannitol salt agar (Himedia) were used and prepared according to manufacturer direction. After obtaining pure colonies and recording important features of the isolated organisms, further identification were done using gram staining and biochemical test with standard methods.
Biochemical test: Gram negative bacteria was identified based on colonial morphology and pigmentation, Oxidase test, Carbohydrate fermentation, H2S production, Citrate utilization, motility, growth at 42˚C, Indole formation, Lysine decarboxylase and Lysine deaminase production, and Urea hydrolysis. Gram positive isolates were also differentiated by gram staining, colonial characteristics, catalase test and coagulase tests.
A standard Kirby-Bauer disk diffusion method was used to determine the antimicrobial susceptibility profiles of the isolates as described by the CLSI, 2014 [
Sample collection, handling, transportation and microbiological analysis and interpretation of results were carried out using standard operating procedures (SOPs). Prior to the actual work Reagents, media and antimicrobial disks were checked for expiry date, damage and storage problems. Laboratory equipment’s were properly cleaned and sterilized before use. Media preparation was made based on the respective manufacturer’s directions. 5% of media per batch/prepared was incubated overnight for sterility check. Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 28753) were used as quality control organisms.
Data was entered and summarized using SPSS version 20 software (USA) and analyzed using the STATA software (StataCorp LP, College. Station, Texas, USA). A p-value of ≤0.05 was considered indicative of a statistically significant difference.
Mean heterotrophic plate count, total coliform count, fecal coliform count and E. coli count were found to be 1.6 × 106 CFU/mL, 2.2 × 106 CFU/100 mL, 2.0 × 105 CFU/100 mL and 1.1 × 104 CFU/100 mL from THWW respectively (
Among the total samples 134 bacterial isolates were detected and 84 (62.7%) were from UHWW and 50 (37.3%) were from THWW. The most frequently isolated bacteria from untreated wastewater samples was Klebsiella spp 14 (16.7%) followed by S. aureus 13 (15.5%) and P. aeruginosa 12 (14.3%), similarly in treated wastewater samples Klebsiella spp 10 (20%), P. aeruginosa 8 (16%) and S. aureus 8 (16%) were frequently isolated (
Indicator organism | UHWW (n = 20) | THWW (n = 20) |
---|---|---|
Total heterotrophic count (CFU/mL) | 1.9 × 108 | 1.6 × 106 |
Total coliform count (CFU/100mL) | 2.6 × 1010 | 2.2 × 106 |
Fecal coliform count (CFU/100mL) | 1.25 × 109 | 2.0 × 105 |
E. coli count (CFU/100mL) | 4.5 × 105 | 1.1 × 104 |
Bacteria isolates | UHWW (n = 20) | THWW (n = 20) | Total (n = 40) |
---|---|---|---|
S. aureus | 13 (15.5%) | 8 (16%) | 21 (15.7%) |
CoNS* | 6 (7.1%) | 5 (10%) | 11 (8.2%) |
Klebsiella spp | 14 (16.7%) | 10 (20%) | 24 (17.9%) |
P. aeruginosa | 12 (14.3%) | 8 (16%) | 20 (14.9%) |
E. coli | 11 (13.1%) | 6 (12%) | 17 (12.7%) |
Salmonella spp | 9 (10.7) | 6 (12%) | 15 (11.2%) |
Shigella spp | 3 (3.6%) | 0 (0) | 3 (2.2% |
Citrobacter spp | 6 (7.1%) | 3 (6%) | 9 (6.7%) |
Enterobacter spp | 4 (4.8%) | 2 (4%) | 6 (4.5%) |
Other isolates | 6 (7.1%) | 2 (4%) | 8 (6%) |
Total | 84 (100%) | 50 (100%) | 134 (100%) |
*CoNS: Coagulase negative staphylococci, other isolates: Acinitobacter spp (only 1 in UHWW), Seratia spp (3 in UHWW and 1 in THWW) and Proteus spp (2 in UHWW and 1 in THWW).
Among bacterial isolates from untreated wastewater, Coagulase negative staphylococci (CoNS) were found to be 100% resistant to Penicillin and 50% to Cefoxitin. S. aureus was also found to be 77% and 38% resistant for Penicillin and Cefoxitin respectively. All isolates of E. coli and Klebsiella spp were 100% resistant to ampicillin (
The total resistance of bacterial isolates from untreated wastewater was higher for penicillin 16/19 (84%) followed by ampicillin 42/52 (81%) and tetracycline 30/72 (42%). However, relatively lower resistance was observed among bacterial isolates to Chloramphenicol 13/71 (18%), gentamycin 19/84 (23%) and ciprofloxacin 20/84 (24%) (
Among bacterial isolates from treated wastewater, all isolates of S. aureus and CoNS were 100% resistant to penicillin. All isolates of E. coli, Klebsiella spp and Citrobacter spp were 100% resistant to ampicillin as shown in the (
The total resistance of isolates from treated wastewater to penicillin was 13/13 (100%) followed by ampicillin 24/29 (83%) and Ceftriaxone 17/29 (59%). Relatively lower resistance among bacterial isolates was observed to ciprofloxacin 10/50 (20%), amikacin 10/50 (20%) and chloramphenicol 10/42 (24%) (
Among isolates from untreated wastewater, 19/72 (26.3%) were resistant for six and more antibiotics (e.g. three isolates of klebsiella spp and one isolates of E. coli were resistant to nine antibiotics; two isolates of klebsiella spp, two isolates of E. coli and one isolates of Citrobacter spp were resistant to eight antibiotics), while 13/84 (15.5%) isolates were resistant for only one antibiotic, and 7/84 (8.3%) were not resistant for any of antibiotics tested (
Bacteria isolates | Antibiotics used N (%) | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
GN | AK | TTC | DO | SXT | CIP | CAF | AMP | CRO | AMC | E | P | CX | ||
S. aureus (13) | R | 2 (15) | 4 (31) | 3 (23) | 1 (8) | 2 (15) | 2 (15) | 4 (31) | NT | NT | NT | 3 (23) | 10 (77) | 5 (38) |
I | 3 (23) | 3 (23) | 4 (31) | 6 (46) | 3 (23) | 2 (15) | 0 (0) | NT | NT | NT | 1 (8) | - | - | |
S | 8 (62) | 6 (46) | 6 (46) | 6 (46) | 8 (62) | 9 (69) | 9 (69) | NT | NT | NT | 9 (69) | 3 (23) | 8 (62) | |
CoNS (6) | R | 0 (0) | 1 (17) | 3 (50) | 2 (33) | 2 (23) | 1 (17) | 1 (17) | NT | NT | NT | 2 (33) | 6 (100) | 3 (50) |
I | 3 (50) | 3 (50) | 2 (23) | 0 (0) | 1 (17) | 2 (23) | 2 (23) | NT | NT | NT | 2 (33) | - | - | |
S | 3 (50) | 2 (33) | 1 (17) | 4 (66) | 3 (50) | 3 (50) | 3 (50) | NT | NT | NT | 2 (33) | 0 (0) | 3 (50) | |
Klebsiella spp (14) | R | 6 (43) | 6 (43) | 9 (64) | 7 (50) | 6 (43) | 4 (29) | 2 (14) | 14 (100) | 6 (43) | 5 (36) | NT | NT | NT |
I | 2 (14) | 3 (21) | 1 (7) | 3 (21) | 2 (14) | 3 (21) | 4 (29) | 0 (0) | 3 (21) | 2 (14) | NT | NT | NT | |
S | 6 (43) | 5 (36) | 4 (29) | 4 (29) | 6 (43) | 7 (50) | 8 (57) | 0 (0) | 5 (36) | 7 (50) | NT | NT | NT | |
E. coli (11) | R | 2 (18) | 2 (18) | 4 (36) | 2 (18) | 5 (45) | 3 (27) | 1 (9) | 11 (100) | 4 (36) | 3 (27) | NT | NT | NT |
I | 3 (27) | 2 (18) | 3 (27) | 3 (27) | 2 (18) | 3 (27) | 2 (18) | 0 (0) | 2 (18) | 2 (18) | NT | NT | NT | |
S | 6 (55) | 7 (64) | 4 (36) | 6 (55) | 4 (36) | 5 (45) | 8 (73) | 0 (0) | 5 (45) | 6 (55) | NT | NT | NT | |
P. aeruginosa (12) | R | 4 (33) | 4 (33) | NT | NT | NT | 3 (25) | NT | NT | NT | NT | NT | NT | NT |
I | 2 (17) | 2 (17) | NT | NT | NT | 4 (33) | NT | NT | NT | NT | NT | NT | NT | |
S | 6 (50) | 6 (50) | NT | NT | NT | 5 (42) | NT | NT | NT | NT | NT | NT | NT | |
Salmonella spp (9) | R | 0 (0) | 1 (11) | 3 (33) | 2 (22) | 1 (11) | 2 (22) | 1 (11) | 5 (56 ( | 3 (33) | 3 (33) | NT | NT | NT |
I | 4 (44) | 2 (22) | 2 (22) | 2 (22) | 3 (33) | 2 (22) | 3 (33) | 2 (22) | 2 (22) | 0 (0) | NT | NT | NT | |
S | 5 (56) | 6 (67) | 4 (44) | 5 (56) | 5 (56) | 5 (56) | 5 (56) | 2 (22) | 4 (44) | 6 (67) | NT | NT | NT | |
Shigella spp (3) | R | 1 (33 | 0 (0) | 0 (0) | 0 (0) | 1 (33) | 1 (33) | 1 (33) | 2 (67) | 2 (67) | 1 (33) | NT | NT | NT |
I | 0 (0) | 1 (33) | 1 (33) | 1 (33) | 1 (33) | 0 (0) | 0 (0) | 0 (0) | 0 | 0 (0) | NT | NT | NT | |
S | 2 (67) | 2 (67) | 2 (67) | 2 (67) | 1 (33) | 2 (67) | 2 (67) | 1 (33) | 1 (33) | 2 (67) | NT | NT | NT | |
Citrobacter spp (6) | R | 2 (33) | 2 (33) | 3 (50) | 2 (33) | 2 (33) | 2 (33) | 1 (17) | 5 (83) | 3 (50) | 1 (17) | NT | NT | NT |
I | 1 (17) | 0 (0) | 1 (17) | 1 (17) | 1 (17) | 0 (0) | 2 (33) | 1 (17) | 1 (17) | 3 (50) | NT | NT | NT | |
S | 3 (50) | 4 (67) | 2 (33) | 3 (50) | 3 (50) | 4 (67) | 3 (50) | 0 (0) | 2 (33) | 2 (33) | NT | NT | NT | |
Enterobacter spp (4) | R | 1 (25) | 1 (25) | 2 (50) | 1 (25 | 1 (25) | 1 (25) | 1 (25) | 2 (50) | 0 (0) | 1 (25) | NT | NT | NT |
I | 2 (50) | 3 (75) | 0 (0) | 2 (50) | 3 (75) | 3 (75) | 0 (0) | 1 (25) | 3 (75) | 2 (50) | NT | NT | NT | |
S | 1 (25) | 0 (0) | 2 (50) | 1 (25) | 0 (0) | 0 (0) | 3 (75) | 1 (25) | 1 (25) | 1 (25) | NT | NT | NT | |
Acinetobacter spp (1) | R | 0 (0) | 0 (0) | 1 (100) | 1 (100) | 1 (100) | 0 (0) | NT | NT | 1 (100) | NT | NT | NT | NT |
I | 1 (100) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (100) | NT | NT | 0 (0) | NT | NT | NT | NT | |
S | 0 (0) | 1 (100 | 0 (0) | 0 (0) | 0 (0) | 0 (0) | NT | NT | 0 (0) | NT | NT | NT | NT | |
Other isolates (5) | R | 1 (20) | 1 (20) | 2 (40) | 1 (20) | 1 (20) | 1 (20) | 1 (20) | 3 (60) | 2 (40) | 2 (40) | NT | NT | NT |
I | 2 (40) | 3 (60) | 1 (20) | 2 (40) | 3 (60) | 3 (60) | 3 (60) | 1 (20) | 0 (0) | 1 (20) | NT | NT | NT | |
S | 2 (40) | 1 (20) | 2 (20) | 2 (40) | 1 (20) | 1 (20) | 1 (20) | 1 (20) | 3 (60) | 2 (40) | NT | NT | NT |
GN: Gentamycin, TTC: Tetracyclin, SXT: Cotrimoxazole, CAF: Chloramphenicol, P: Penicillin, AK: Amikacin, DO: Doxycycline, CIP: Ciprofloxacin, E: Erytromyacin, CX: Cefoxitin, AMC: Amoxacillin-clavulunic acid, AMP: Ampicillin, CRO: Ceftriaxone, R: Resistance, S: Susceptible, I, Intermediate, NT: Not Tested, Other isolates: Seratia spp (3) and Proteus spp (2).
Bacteria isolates | Antibiotics used N (%) | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
GN | AK | TTC | DO | SXT | CIP | CAF | AMP | CRO | AMC | E | P | CX | ||
S. aureus (8) | R | 2 (25) | 1 (13) | 2 (25) | 3 (38) | 1 (13) | 1 (13) | 2 (25) | NT | NT | NT | 3 (38) | 8 (100) | 3 (38) |
I | 1 (13) | 4 (50) | 3 (38) | 2 (25) | 4 (50) | 3 (38) | 1 (13) | NT | NT | NT | 1 (13) | - | - | |
S | 5 (63) | 3 (38) | 3 (38) | 3 (38) | 3 (38) | 4 (50) | 5 (63) | NT | NT | NT | 4 (50) | 0 (0) | 5 (63) | |
CoNS (5) | R | 1 (20) | 0 (0) | 2 (40) | 2 (40) | 1 (20) | 0 (0) | 2 (40) | NT | NT | NT | 1 (20) | 5 (100) | 2 (40) |
I | 3 (60) | 2 (40) | 0 (0) | 0 (0) | 0 (0) | 2 (40) | 0 (0) | NT | NT | NT | 3 (60) | - | - | |
S | 1 (20) | 3 (60) | 3 (60) | 3 (60) | 4 (80) | 3 (60) | 3 (60) | NT | NT | NT | 1 (20) | 0 (0) | 3 (60) | |
Klebsiella spp (10) | R | 6 (60) | 4 (40) | 4 (40) | 4 (40) | 6 (60) | 2 (20) | 2 (20) | 10 (100) | 5 (50) | 3 (30) | NT | NT | NT |
I | 0 (0) | 1 (10) | 2 (20) | 3 (30) | 0 (0) | 4 (40) | 3 (30) | 0 (0) | 1 (10) | 3 (30) | NT | NT | NT | |
S | 4 (40) | 5 (50) | 4 (40) | 3 (30) | 4 (40) | 4 (40) | 5 (50) | 0 (0) | 4 (40) | 4 (40) | NT | NT | NT | |
E. coli (6) | R | 2 (33) | 2 (33) | 2 (33) | 2 (33) | 4 (67) | 2 (33) | 1 (17) | 6 (100) | 3 (50) | 3 (50 | NT | NT | NT |
I | 1 (17) | 0 (0) | 0 (0) | 1 (17) | 0 (0) | 1 (17) | 1 (17) | 0 (0) | 2 (33) | 1 (17) | NT | NT | NT | |
S | 3 (50) | 4 (67) | 4 (67) | 3 (50) | 2 (33) | 3 (50) | 4 (67) | 0 (0) | 1 (17) | 2 (33) | NT | NT | NT | |
P. aeruginosa (8) | R | 4 (50) | 2 (25) | NT | NT | NT | 3 (38) | NT | NT | NT | NT | NT | NT | NT |
I | 2 (25) | 4 (50) | NT | NT | NT | 0 (0) | NT | NT | NT | NT | NT | NT | NT | |
S | 2 (25) | 2 (25) | NT | NT | NT | 5 (62) | NT | NT | NT | NT | NT | NT | NT | |
Salmonella spp (6) | R | 2 (33) | 0 (0) | 3 (50) | 2 (33) | 2 (33) | 1 (17) | 1 (17) | 3 (50) | 4 (67) | 2 (33) | NT | NT | NT |
I | 1 (17) | 2 (33) | 0 (0) | 0 (0) | 1 (17) | 2 (33) | 2 (33) | 0 (0) | 0 (0) | 1 (17) | NT | NT | NT | |
S | 3 (50) | 4 (67) | 3 (50) | 4 (67) | 3 (50) | 3 (50) | 3 (50) | 3 (50) | 2 (33) | 3 (50) | NT | NT | NT | |
Citrobacter spp (3) | R | 1 (33) | 1 (33) | 3 (100) | 2 (67) | 1 (33) | 1 (33) | 1 (33) | 3 (100) | 2 (67) | 2 (67) | NT | NT | NT |
I | 2 (67) | 0 (0) | 0 (0) | 0 (0) | 2 (67) | 0 (0) | 0 | 0 (0) | 1 (33) | 0 (0) | NT | NT | NT | |
S | 0 (0) | 2 (67) | 0 (0) | 1 (33) | 0 (0) | 2 (67) | 2 (67) | 0 (0) | 0 (0) | 1 (33) | NT | NT | NT | |
Enterobacter spp (2) | R | 0 (0) | 0 (0) | 1 (50) | 1 (50) | 1 (50) | 0 (0) | 0 | 1 (50) | 1 (50) | 1 (50 | NT | NT | NT |
I | 2 (100) | 2 (100) | 1 (50) | 0 (0) | 0 (0) | 2 (100) | 1 (50) | 1 (50) | 0 (0) | 1 (50) | NT | NT | NT | |
S | 0 (0) | 0 (0) | 0 (0) | 1 (50) | 1 (50) | 0 (0) | 1 (50) | 0 (0) | 1 (50) | 0 (0) | NT | NT | NT | |
Other isolates (2) | R | 1 (50) | 0 (0) | 2 (100) | 1 (50) | 1 (50) | 0 (0) | 1 (50) | 1 (50) | 2 (100) | 2 (100) | NT | NT | NT |
I | 1 (50) | 1 (50) | 0 (0) | 0 (0) | 1 (50) | 2 (100) | 0 | 1 (50) | 0 (0) | 0 (0) | NT | NT | NT | |
S | 0 (0) | 1 (50) | 0 (0) | 1 (50) | 0 (0) | 0 (0) | 1 (50) | 0 (0) | 0 (0) | 0 (0) | NT | NT | NT |
GN: Gentamycin, TTC: Tetracyclin, SXT: Cotrimoxazole, CAF: Chloramphenicol, P: Penicillin, AK: Amikacin, DO: Doxycycline, CIP: Ciprofloxacin, E: Erytromyacin, CX: Cefoxitin, AMC: Amoxacillin-clavulunic acid, AMP: Ampicillin, CRO: Ceftriaxone R: Resistance, S: Susceptible, I, Intermediate, NT: Not Tested, Other isolates: Seratia spp (1) and Proteus spp (1).
Bacteria isolates | Number of isolates | R0 | R1 | R2 | R3 | R4 | R5 | ≥R6 |
---|---|---|---|---|---|---|---|---|
S. aureus | 13 | 0 (0) | 3 (23.1) | 4 (30.8) | 3 (23.1) | 0 (0) | 1 (7.7) | 2 (15.4) |
CoNS | 6 | 0 (0) | 0 (0) | 3 (50) | 1 (16.7) | 1 (16.7) | 0 (0) | 1 (16.7) |
Klebsiella spp | 14 | 0 (0) | 1 (7.1) | 3 (21.4) | 1 (7.1) | 0 (0) | 1 (7.1) | 8 (57.1) |
E. coli | 11 | 0 (0) | 2 (18.2) | 2 (18.2) | 1 (9.1) | 1 (9.1) | 2 (18.2) | 3 (27.3) |
P. aeroginosa | 12 | 5 (41.7) | 2 (18.2) | 3 (25) | 2 (18.2) | NT | NT | NT |
Salmonella spp | 9 | 1 (11.1) | 0 (0) | 2 (22.2) | 1 (11.1) | 1 (11.1) | 2 (22.2) | 2 (22.2) |
Shigella spp | 3 | 1 (33.3) | 1 (33.3) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (33.3) |
Citrobacter spp | 6 | 0 (0) | 2 (33.3) | 0 (0) | 0 (0) | 0 (0) | 2 (33.3) | 2 (33.3) |
Entrobacter spp | 4 | 0 (0) | 2 (50.0) | 1 (25) | 0 (0) | 1 (25) | 0 (0) | 0 (0) |
Other isolates | 6 | 0 (0) | 0 (0) | 2 (33.3) | 1 (16.7) | 2 (66.7) | 1 (016.7) | 0 (0) |
Total | 84 | 7 (8.3) | 13 (15.5) | 20 (23.8) | 10 (11.9) | 6 (8.3) | 9 (12.5) | 19 (26.4) |
R0: Not resistant for any antibiotics tested, R1: Resistant to one antibiotic, R2: Resistant to two antibiotics, R3: Resistant to three antibiotics, R4: Resistant to four antibiotics, R5: Resistant to five antibiotics, ≥R6: Resistant to six or more antibiotics, NT: Not Tested, Other isolates: Accinitobacter spp (1), Seratia spp (3) and Proteus spp (2).
Among isolates from treated wastewater, 12/42 (28.6%) were resistant for six and more antibiotics (e.g. two isolates of Klebsiella spp and one isolates of CoNS were resistant for ten antibiotics, one isolates of Citrobacter spp were also resistant for nine antibiotics), while 8/50 (16%) were resistant for only one antibiotics and there was no isolates that are not resistant for any of antibiotics tested (
Among isolates from both treated and untreated hospital wastewater the overall prevalence of multi-drug resistance (resistant to two and above antibiotics) in this study was found to be 106/134 (79.1%). Multi-drug resistance in untreated wastewater sample was found to be 64/84 (76.2%) whereas in treated wastewater was found to be 42/50 (84%). In this study isolates from treated wastewater was found to be 1.64 times resistant for many drug than isolates from untreated hospital wastewater (COR:1.64, 95% CI: 1.15 - 3.29, P: 0.001).
The present study showed that mean heterotrophic plate count (1.6 × 106 CFU/mL), was exceeded the permissible limit of Environment Protection Agency, EPA [
Bacteria isolates | Number of isolates | R0 | R1 | R2 | R3 | R4 | R5 | ≥R6 |
---|---|---|---|---|---|---|---|---|
S. aureus | 8 | 0 (0) | 1 (12.5) | 2 (25) | 2 (25) | 1 (12.5) | 0 (0) | 2 (25) |
CoNS | 5 | 0 (0) | 0 (0) | 0 (0) | 1 (20) | 2 (40) | 1 (20) | 1 (20) |
Klebsiella spp | 10 | 0 (0) | 1 (10) | 2 (20) | 2 (20) | 1 (10) | 1 (10) | 3 (30) |
E. coli | 6 | 0 (0) | 1 (16.7) | 0 (0) | 1 (16.7) | 2 (33.3) | 0 (0) | 2 (33.3) |
P. aeroginosa | 8 | 0 (0) | 5 (62.5) | 1 (12.5) | 2 (25) | NT | NT | NT |
Salmonella spp | 6 | 0 (0) | 0 (0) | 1 (16.7) | 2 (33.3) | 1 (16.7) | 0 (0) | 2 (33.3) |
Citrobacter spp | 3 | 0 (0) | 0 (0) | 1 (33.3) | 0 (0) | 0 (0) | 0 (0) | 2 (66.7) |
Entrobacter spp | 2 | 0 (0) | 0 (0) | 1 (50) | 0 (0) | 0 (0) | 1 (50) | 0 (0) |
Other isolates | 2 | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (50) | 1 (50) | 0 (0) |
Total | 50 | 0 (0) | 8 (16) | 8 (16) | 10 (20) | 8 (19.0) | 4 (9.5) | 12 (28.6) |
R0: Not resistant for any antibiotics tested, R1: Resistant to one antibiotic, R2: Resistant to two antibiotics, R3: Resistant to three antibiotics, R4: Resistant to four antibiotics, R5: Resistant to five antibiotics, ≥R6: Resistant to six or more antibiotics, NT: Not Tested, Other isolates: Seratia spp (1) and Proteus spp (1).
Microbial contamination of treated hospital wastewater was 2.0 × 105 of fecal coliforms, which was higher than WHO maximum tolerable limit for fecal indicator bacteria (≤103/100 mL for unrestricted irrigation and ≤105/100 mL for restricted irrigation) [
In the present study bacterial isolates such as Klebsiella spp 10 (20%), P. aeruginosa 8 (16%), S. aureus 8 (16%), E. coli 7 (14%) and salmonella spp 6 (12%) were also frequently detected in treated wastewater. The same study conducted in Ethiopia, Hawassa University Referral Hospital, reported Salmonella spp, Shigella spp, E. coli and S. aureus from hospital effluent [
Among bacterial isolates from treated wastewater, S. aureus and CoNS were 100% resistant to penicillin and also E. coli, Klebsiella spp and Citrobacter spp were 100% resistant to ampicillin. In general most bacterial isolates were highly resistant to tetracycline, doxycycline, cotrimoxazole, amoxicillin-clavulinic acid and ceftriaxone. The same study in India showed simultaneous resistance of isolates for ampicillin, ampicillin with clavulinic acid, cotrimoxazole, tetracycline, first, second and third generation cephalosporins in the final effluent of wastewater treatment plant [
Overall resistance of bacterial isolates from THWW for methicillin was found to be 38%. Methicillin resistant Staphylococci (MRSA and MRCoNS) were not studied well in our country from hospital effluents. But as indicated by Abulreesh [
This study also observed that, most bacterial isolates from the THWW shows higher rate of resistance than bacterial isolates from the UHWW and shows resistant bacteria isolates were able to survive the journey to the inlet of sewage treatment plant and treatment process. The same result was reported from Alice, Eastern Cape province of South Africa [
High percentages of multi-drug resistance for the majority of the isolates in THWW were discharged to the environment. This was supported by Study conducted in Switzerland which showed that increased proportions of highly and extremely multi-resistant bacteria among the isolated sulfamethoxazole/ trimethoprim and streptomycin resistant strains in the sample of treated wastewater compared to the wastewater treatment plant inlet sample [
The release of MDR bacteria to the environment causes extensive genetic exchange; where opportunistic pathogens (commonly found in free-living communities) may become resistant upon acquiring resistance mechanisms. Therefore, reduction of selective pressure by regulating the use of antibiotics is a key step to undermine the spread of resistance in hospital wastewater in order not to favored resistant strains [
In the present study, high numbers of indicator organisms were obtained from treated hospital wastewater, which exceeded the WHO, HPA, EPA and FAO standard permissible levels. Significant pathogenic and potentially pathogenic bacteria were also isolated from the treated wastewater. There was high prevalence of drug resistant isolates from untreated and treated hospital wastewater suggesting their persistence in the hospital environment, and their ability to pass the processes of treatment plant. Therefore Liquid waste treatment system (Chlorination) should be developed to disinfect pathogens in treated wastewater effluents.
Approved.
The authors declare that they have no competing interests.
TA participated in its design and performed the laboratory activities. TA analyzed the data and wrote the manuscript. LN, AK and YW reviewed the manuscript. All authors read and approved the final manuscript.
Asfaw, T., Negash, L., Kahsay, A. and Weldu, Y. (2017) Antibiotic Resistant Bacteria from Treated and Untreated Hospital Wastewater at Ayder Referral Hospital, Mekelle, North Ethiopia. Advances in Microbiology, 7, 871-886. https://doi.org/10.4236/aim.2017.712067