Advances in Microbiology, 2017, 7, 871-886

http://www.scirp.org/journal/aim
ISSN Online: 2165-3410
ISSN Print: 2165-3402

Antibiotic Resistant Bacteria from

Treated and Untreated Hospital
Wastewater at Ayder Referral Hospital,
Mekelle, North Ethiopia

Tsegahun Asfaw1,2, Letemichael Negash2, Amlsha Kahsay2, Yemane Weldu2

Department of Medical Laboratory Science, Medical Microbiology Unit, DebreBerhan University, Debre Berhan, Ethiopia
1

Department of Medical Microbiology and Immunology, Institute of Biomedical Sciences, Mekelle University, Mekelle, Ethiopia
2

How to cite this paper: Asfaw, T., Negash, Abstract

L., Kahsay, A. and Weldu, Y. (2017) Anti-
biotic Resistant Bacteria from Treated and The widespread emergence of antibiotic resistance among bacterial pathogens
Untreated Hospital Wastewater at Ayder has become one of the most serious challenges in Ethiopia. This study deter-
Referral Hospital, Mekelle, North Ethiopia.
mined the prevalence and drug resistance patterns of bacterial pathogens iso-
Advances in Microbiology, 7, 871-886.
https://doi.org/10.4236/aim.2017.712067 lated from treated and untreated wastewater released from Ayder Referral
Hospital in Northern Ethiopia. A cross sectional study design was conducted
Received: November 9, 2017 from September-December, 2015 in wastewater released from Ayder referral
Accepted: December 24, 2017
hospital. A total of 40 composite samples were aseptically collected, trans-
Published: December 27, 2017
ported and processed for enumeration of indicator organisms, bacteriological
Copyright © 2017 by authors and identification and susceptibility testing following standard procedure. Data
Scientific Research Publishing Inc.
obtained were entered and analyzed using SPSS version 20. Mean hetero-
This work is licensed under the Creative
Commons Attribution International trophic plate count, total coliform count, fecal coliform count and E. coli
License (CC BY 4.0). count were found to be 1.6 × 106 CFU/mL, 2.2 × 106 CFU/100 mL, 2.0 × 105
http://creativecommons.org/licenses/by/4.0/ CFU/100 mL and 1.1 × 104 CFU/100 mL from treated wastewater respectively.
Open Access Among the total samples 134 bacterial isolates were detected and [84 (62.7%)]
were from untreated wastewater and [50 (37.3%)] were from treated waste-
water. The most frequently isolated bacteria from untreated wastewater sam-
ples 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 fre-
quently 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 con-
cluded that treated hospital wastewater contains large numbers of antibiotic

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T. Asfaw et al.

resistant bacteria. Therefore, there should be continuous monitoring and

evaluation of the effluent quality of the ponds and chlorination of the final ef-
fluent should be developed.

Keywords
Indicator Organism, Bacterial Isolates, Drug Resistance,
Treated Wastewater, Untreated Wastewater

1. Introduction
Wastewater is an ideal media for a wide range of microorganisms especially bac-
teria, viruses and protozoa, and used as a reservoir for resistant bacteria [1]. It
carries the resistant bacteria introduced into the sewage system that come from
human excretions, liquid waste discharged from domestic home, agricultural
and commercial sectors, pharmaceutical and hospitals [2] [3].
The volume of antibiotics used in hospitals released into effluent results a se-
lection 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 [4]. Accordingly, hospital wastewater
could increase the numbers of resistant bacteria in the recipient sewers by both
mechanisms of introduction and selection pressure [5]. The release of resistant
bacteria to the receiving environment can pose public health impact through,
carrying transmissible gene, by acting as a vector or reservoir of resistant gene
[6] [7]. The most common bacterial pathogens found in hospital wastewater are
Salmonella, Shigella, S. aureus, P. aeruginosa, Vibrio, Clostridium, Yersinia,
Campylobacter, Leptospira and groups of total coliforms consisting of Serratia,
Enterobacter, Klebsiella, and Escherichia coli (E. coli) [8] [9]. Those bacterial
pathogens are among of the most dangerous contaminants on human health,
and then the effluents from hospital wastewaters are one of the most serious
pollutants discharging to the environment [9] [10]. The multidrug resistance
patterns seen in the bacterial isolates from hospital effluent samples include
most of the antibiotics used presently for treating human infections. The worst
fear apprehended is the transfer of such resistance to bacterial pathogens causing
infections in the community. In that case most of the presently available antibio-
tics will be vain against the infectious organisms. The origin of such multidrug
resistance bacterial strains appears to be the hospital environment due to selec-
tive pressure [11]. Dissemination of antimicrobial resistance bacteria in the en-
vironment is a major problem in developing countries, mainly due to improper
antibiotic usage, ineffective infection control program and lack of better man-
agement of hospital wastewater. In Ethiopia, rapid urbanization and industriali-
zation with improper environmental planning often lead to discharge of indus-
trial and hospital sewage effluent directly to the environment also a problem [12]
[13].

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 T. Asfaw et al.

2. Materials and Methods

2.1. Study Area
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 de-
signed 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 faculta-
tive pond and its primary function is removal of pathogenic microorganisms.
The last step in treatment process is release of treated effluent to open field con-
tinuously 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.

2.2. Study Design and Period

Cross sectional study design was conducted to collect hospital wastewater sam-
ples from September, 2015 to December, 2015.

2.3. Sampling Frequency and Sampling Technique

A “Composite-sampling” technique was applied to collect the most representa-
tive samples according to guidelines of wastewater sampling techniques stated
on APHA [14]. A total of 80 partial samples were collected from both sampling
site with 4 hour interval in the study period and finally 40 composite samples
were processed for bacteriological analysis, identification and susceptibility test-
ing. Partial samples were collected two times a day with four hour interval from
both sites in 125 mL cleaned and sterile microbiological glass bottles. The first
samples were collected in the morning 10 AM from UHWW sampling site and
10:30 AM from THWW sampling site and transported immediately after collec-
tion and stored in refrigerator at 4˚C. After collection of afternoon samples 2
PM from UHWW and 2:30 PM from THWW, the partial samples were pooled
(untreated to untreated and treated to treated) aseptically to 250 mL sized
cleaned and sterilized microbiological glass bottle. Then it was stored in refrige-
rator at 4˚C until the samples is processed.

2.4. Bacteriological Analysis of Wastewater

Total heterotrophic plate count: Duplicate plates were prepared for each vo-
lume of sample examined. All the samples were vigorously shaken before prepa-
ration of dilutions then serial 10-fold dilutions of samples were prepared in phy-
siological saline, and 1 mL aliquot was spread over nutrient agar plate (Himedia).

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T. Asfaw et al.

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. Final-
ly bacterial load were estimated using MPN table and dilution factor. The bac-
terial 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. Fur-
thermore 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.

2.5. Isolation and Identification of Bacteria

Culture technique: The sample was investigated for further isolation and iden-
tification of bacterial pathogens. Representative diluted sample of 1 ml aliquots
was plated on selective and differential media. MacConkey agar (SRL-sisco re-
search 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 organ-
isms, 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, Ly-
sine decarboxylase and Lysine deaminase production, and Urea hydrolysis.
Gram positive isolates were also differentiated by gram staining, colonial cha-
racteristics, catalase test and coagulase tests.

2.6. Antimicrobial Susceptibility Testing

A standard Kirby-Bauer disk diffusion method was used to determine the anti-
microbial susceptibility profiles of the isolates as described by the CLSI, 2014
[15]. Bacterial isolates were tested for the following commonly prescribed drug:
Erythromycin (15 μg), Gentamyacin (10 μg), Amikacin (30 μg), Amoxicil-
lin-Clavulunic acid (30 μg), Ceftriaxone (30 μg), Ciprofloxacin (5 μg), Tetracyc-
line (30 μg), Cotrimoxazole (25 μg), Ampicillin (10 μg), Penicillin (10 μg),
Chloromaphenicol (30 μg), Doxycycline (30 μg) and Cefoxitin (30 μg). Interpre-
tation was made using susceptibility breakpoints of CLSI, 2014 and diameter of

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 T. Asfaw et al.

the zone of inhibition around the disc was measured to the nearest millimeter
using a metal caliper and the isolates were classified as sensitive, intermediate
and resistant.

2.7. Data Quality Assurance

Sample collection, handling, transportation and microbiological analysis and in-
terpretation 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), Sta-
phylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 28753)
were used as quality control organisms.

2.8. Data Management and Statistical Analysis

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.

3. Results
3.1. Bacteriological Analysis of Wastewater before
and after Treatment
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 (Table 1).

3.2. Bacterial Isolates

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 (Table 2).

Table 1. Mean of indicator organisms in treated and untreated wastewater released from
Ayder referral hospital, Mekelle, North Ethiopia (September-December 2015).

Indicator organism UHWW (n = 20) THWW (n = 20)

Total heterotrophic count (CFU/mL) 1.9 × 10 8
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

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T. Asfaw et al.

Table 2. Frequency of bacterial isolates from treated and untreated wastewater released
from Ayder referral hospital, Mekelle, North Ethiopia (September-December 2015).

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).

3.3. Drug Susceptibility Profile of Bacterial Isolates

Among bacterial isolates from untreated wastewater, Coagulase negative sta-
phylococci (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 (Table 3).
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 ciprof-
loxacin 20/84 (24%) (Figure 1).
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 (Table 4).
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%). Rela-
tively lower resistance among bacterial isolates was observed to ciprofloxacin
10/50 (20%), amikacin 10/50 (20%) and chloramphenicol 10/42 (24%) (Figure
2).
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 (Table 5).

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 T. Asfaw et al.

Table 3. Drug Susceptibility profile of bacterial isolates from untreated wastewater released from Ayder referral hospital, Mekelle,
North Ethiopia (September-December 2015).

Antibiotics used N (%)

Bacteria isolates
GN AK TTC DO SXT CIP CAF AMP CRO AMC E P CX

R 2 (15) 4 (31) 3 (23) 1 (8) 2 (15) 2 (15) 4 (31) NT NT NT 3 (23) 10 (77) 5 (38)

S. aureus (13) 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)

R 0 (0) 1 (17) 3 (50) 2 (33) 2 (23) 1 (17) 1 (17) NT NT NT 2 (33) 6 (100) 3 (50)

CoNS (6) 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)

R 6 (43) 6 (43) 9 (64) 7 (50) 6 (43) 4 (29) 2 (14) 14 (100) 6 (43) 5 (36) NT NT NT

Klebsiella spp (14) 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

R 2 (18) 2 (18) 4 (36) 2 (18) 5 (45) 3 (27) 1 (9) 11 (100) 4 (36) 3 (27) NT NT NT

E. coli (11) 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

R 4 (33) 4 (33) NT NT NT 3 (25) NT NT NT NT NT NT NT

P. aeruginosa (12) 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

R 0 (0) 1 (11) 3 (33) 2 (22) 1 (11) 2 (22) 1 (11) 5 (56 ( 3 (33) 3 (33) NT NT NT

Salmonella spp (9) 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

R 1 (33 0 (0) 0 (0) 0 (0) 1 (33) 1 (33) 1 (33) 2 (67) 2 (67) 1 (33) NT NT NT

Shigella spp (3) 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

R 2 (33) 2 (33) 3 (50) 2 (33) 2 (33) 2 (33) 1 (17) 5 (83) 3 (50) 1 (17) NT NT NT

Citrobacter spp (6) 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

R 1 (25) 1 (25) 2 (50) 1 (25 1 (25) 1 (25) 1 (25) 2 (50) 0 (0) 1 (25) NT NT NT

Enterobacter spp (4) 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

R 0 (0) 0 (0) 1 (100) 1 (100) 1 (100) 0 (0) NT NT 1 (100) NT NT NT NT

Acinetobacter spp (1) 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
R 1 (20) 1 (20) 2 (40) 1 (20) 1 (20) 1 (20) 1 (20) 3 (60) 2 (40) 2 (40) NT NT NT

Other isolates (5) 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).

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Table 4. Drug Susceptibility profile of bacterial isolates from treated wastewater released from Ayder referral hospital, Mekelle,
North Ethiopia (September-December 2015).

Antibiotics used N (%)

Bacteria isolates
GN AK TTC DO SXT CIP CAF AMP CRO AMC E P CX

R 2 (25) 1 (13) 2 (25) 3 (38) 1 (13) 1 (13) 2 (25) NT NT NT 3 (38) 8 (100) 3 (38)

S. aureus (8) 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)

R 1 (20) 0 (0) 2 (40) 2 (40) 1 (20) 0 (0) 2 (40) NT NT NT 1 (20) 5 (100) 2 (40)

CoNS (5) 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)

R 6 (60) 4 (40) 4 (40) 4 (40) 6 (60) 2 (20) 2 (20) 10 (100) 5 (50) 3 (30) NT NT NT

Klebsiella spp (10) 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

R 2 (33) 2 (33) 2 (33) 2 (33) 4 (67) 2 (33) 1 (17) 6 (100) 3 (50) 3 (50 NT NT NT

E. coli (6) 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

R 4 (50) 2 (25) NT NT NT 3 (38) NT NT NT NT NT NT NT

P. aeruginosa (8) 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

R 2 (33) 0 (0) 3 (50) 2 (33) 2 (33) 1 (17) 1 (17) 3 (50) 4 (67) 2 (33) NT NT NT

Salmonella spp (6) 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

R 1 (33) 1 (33) 3 (100) 2 (67) 1 (33) 1 (33) 1 (33) 3 (100) 2 (67) 2 (67) NT NT NT

Citrobacter spp (3) 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

R 0 (0) 0 (0) 1 (50) 1 (50) 1 (50) 0 (0) 0 1 (50) 1 (50) 1 (50 NT NT NT

Enterobacter spp (2) 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

R 1 (50) 0 (0) 2 (100) 1 (50) 1 (50) 0 (0) 1 (50) 1 (50) 2 (100) 2 (100) NT NT NT

Other isolates (2) 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).

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 T. Asfaw et al.

90

80
% Resistance/ Intermidite/Susceptible

70

60

50 Resistance
Intermidiate
40
Susceptible
30

20

10

0
GN AK TTC DO SXT CIP CAF AMP CRO AMC E P CX
GN: Gentamycin, AK: Amikacin, TTC: Tetracyclin, Do: Doxycyclin, SXT: Cotrimoxazole,
CIP: Ciprofloxacin, CAF: Chloramphenicol, AMP: Ampicillin, CRO: Ceftriazone, AMC:
Amoxacillin-clavulunic acid, E: Erytromyacin, P: Penicillin, CX: cefoxitin
Figure 1. Total drug susceptibility profile of isolates from untreated wastewater released from Ayder referral hospital, Mekelle,
North Ethiopia (September-December 2015).

120

100
% Resistance/Intermidiate/

80
Resistance
susceptible

Intermidiate
60
Susceptible

40

20

0
GN AK TTC DO SXT CIP CAF AMP CRO AMC E P CX
GN: Gentmycin, AK: Amikacin: TTC: Tetracycline, DO: Doxycyclin, SXT: Cotrimoxazole,
CIP: Ciprofloxacin, CAF: Chloramphenicol, AMP: Ampicillin, CRO: Ceftriazone, AMC:
amoxacillin- clavulunic acid, E: Erytromyacin, P: Penicillin, CX: Cefoxitin
Figure 2. Total drug susceptibility profile of isolates from treated wastewater released from Ayder referral hospital, Mekelle, North
Ethiopia (September-December 2015).

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T. Asfaw et al.

Table 5. Multi-drug resistance patterns of bacterial isolates from untreated wastewater

released from Ayder Referral Hospital, Mekelle, North Ethiopia (September-December
2015).

Number
Bacteria isolates R0 R1 R2 R3 R4 R5 ≥R6
of isolates

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: Re-
sistant 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 resis-
tant 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
(Table 6).
Among isolates from both treated and untreated hospital wastewater the over-
all 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 hos-
pital wastewater (COR:1.64, 95% CI: 1.15 - 3.29, P: 0.001).

4. Discussion
The present study showed that mean heterotrophic plate count (1.6 × 106
CFU/mL), was exceeded the permissible limit of Environment Protection
Agency, EPA [16] and Health Protection Agency, HPA [17] (<1000 CFU/mL)
for treated wastewater. Total coliform count (2.2 × 106 CFU/100 mL) also failed
to fulfill the requirements of the revised guidelines on the quality of treated
wastewater used in agriculture, in public parks (<5 × 103 CFU/100mL) [18] [19].

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 T. Asfaw et al.

Table 6. Multi-drug resistance patterns of bacterial isolates from treated wastewater

released from Ayder Referral Hospital, Mekelle, North Ethiopia (September-December
2015).

Number
Bacteria isolates R0 R1 R2 R3 R4 R5 ≥R6
of isolates

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: Re-
sistant 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 co-
liforms, which was higher than WHO maximum tolerable limit for fecal indica-
tor bacteria (≤103/100 mL for unrestricted irrigation and ≤105/100 mL for re-
stricted irrigation) [20] [21]. Faecal coliform count was also higher than FAO
standard allowable limit of 5000 CFU/100 mL [22]. High density of E. coli (1.1 ×
104) in treated wastewater was also an indication of fecal pollution of environ-
ment due to human activities.
In the present study bacterial isolates such as Klebsiella spp 10 (20%), P. aeru-
ginosa 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 [3]. Similarly, from
Thailand pathogenic bacteria like Vibrio spp and Salmonella spp as well as other
potentially pathogenic bacteria were reported [23]. Likewise, study in India
showed large numbers of enteric-bacteria, S. aureus and P. aeruginosa [24].
Study in Tunisia also showed large amount of pathogenic bacteria like Salmo-
nella that have a real impact in human health [25]. The highest prevalence of
Salmonella species is due to the fact that it is highly associated with typhoid fev-
er, paratyphoid fever and gastroenteritis, especially of developing countries. Dif-
ferent research also revealed that its survival in fresh water and sewage is less
than 60 days and better recovered from sewage system compared to Shigella.
The highest microbial contamination of treated wastewater shows wastewater
treatment plant may not be efficient with regard to removing bacteria in the final
effluent that is discharged to the receiving environment.

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T. Asfaw et al.

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 iso-
lates for ampicillin, ampicillin with clavulinic acid, cotrimoxazole, tetracycline,
first, second and third generation cephalosporins in the final effluent of waste-
water treatment plant [26]. Study in Alexandria, Egypt also showed the presence
of antibiotic resistant extended spectrum B-lactamase (ESBL) producing bacteria
at the end of wastewater purification process [2], posing a risk of its spread to
the environment and subsequent human and animal exposure.
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 Abu-
lreesh [27] from International Conference on biotechnology and environment
management, Singapore multidrug resistant staphylococci (S. aureus and coagu-
lase negative Staphylococci) had been a common problem and recovered from
diverse environmental sources, such as drinking water supplies and hospital en-
vironments which warns public health concern since contamination of river and
lake with this pathogen may pose risk to the public health associated with Sta-
phylococcal infection and food poisoning.
This study also observed that, most bacterial isolates from the THWW shows
higher rate of resistance than bacterial isolates from the UHWW and shows re-
sistant 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 [28] and European countries [29] [30],
where higher rate of resistance in bacterial isolates from final effluent of waste-
water treatment plant was found. Presences of high percentage of drug resistant
isolates from the final effluent of WWTP suggest that, hospital wastewater could
have contributed massively to the resistances observed among the isolates from
the final effluent. These can be due to the fact that, hospital wastewater contains
a diverse group of pathogenic, commensal and environmental bacteria. This
characteristic composition makes sewage particularly suitable ecological niche
for the growth and spread of antibiotic resistance due to selection pressure and
horizontal gene transfer [31] [32] [33].
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 waste-
water compared to the wastewater treatment plant inlet sample [34]. Study con-
ducted in Australia [35], Brazil [36] and China [37] that investigates a hospital
sewage treatment system also showed, the treatment of the hospital wastewater
may not be totally effective in removing multiple drug resistant bacteria and

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 T. Asfaw et al.

resistant gene from hospital wastewater.

The release of MDR bacteria to the environment causes extensive genetic ex-
change; where opportunistic pathogens (commonly found in free-living com-
munities) may become resistant upon acquiring resistance mechanisms. There-
fore, 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 [38].

5. Conclusion
In the present study, high numbers of indicator organisms were obtained from
treated hospital wastewater, which exceeded the WHO, HPA, EPA and FAO stan-
dard permissible levels. Significant pathogenic and potentially pathogenic bacte-
ria 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.

Ethical Approval
Approved.

Competing Interests
The authors declare that they have no competing interests.

Author Contributions
TA participated in its design and performed the laboratory activities. TA ana-
lyzed the data and wrote the manuscript. LN, AK and YW reviewed the manu-
script. All authors read and approved the final manuscript.

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