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Nigerian Journal of Microbiology, Vol. 25: 2243 – 2250 2011 ANTIBIOTIC RESISTANCE PROFILE OF WASTEWATER ISOLATES OBTAINED
Akubuenyi F.C. 1, G.E. Arikpo1, C.J. Ogugbue2, J.F. Mfongeh1, E. V. Akpanumun1.
1 Department of Biological Sciences, Cross River University of Technology, Cross River State, Nigeria.
2 Department of Microbiology, University of Port Harcourt, Rivers State, Nigeria.
The antibiotic resistance profile of bacterial isolates obtained from the wastewaters of the
University of Calabar Teaching Hospital (UCTH) and the General Hospital Calabar (GHC),
Cross River State, Nigeria, was determined using the disc-diffusion method. A total of 125
bacterial isolates from both hospitals wastewater comprising of the following genera:
Escherichia; Salmonella; Shigella; Klebsiella; Pseudomonas; Streptococcus; Bacillus;
Staphylococcus and Proteus, were tested for their antibiotic resistance capability. Data
obtained showed that all the isolates from both hospitals had multiple antibiotic resistance
(MAR). Over fifty five percent of the isolates from UCTH and 12.5% of the isolates from
GHC exhibited resistance to the antibiotics tested (amoxycilin, gentamycin, augumetin,
cotrimoxazole). Amongst the UCTH isolates, 5 different antibiotic patterns were observed
ranging from 6-12 MAR combinations while 8 different antibiotic resistance patterns ranging
from 4-12 MAR combinations were obtained from the GHC isolates. All the UCTH isolates
were resistant to the antibiotics commonly used in the hospital (amoxicillin, augumentin,
chloramphenicol, gentamycin, erythromycin, tetracycline, ciprofloxacin, streptomycin and
cotrimaxazole) except Escherichia coli and Pseudomonas which are sensitive to
ciprofloxacin. The same trend was obtained for the GHC isolates for the commonly used
antibiotics (chloramphenicol, erythromycin, tetracycline, streptomycin, cotrimaxazole) in
GHC. The lowest resistance of 25% was obtained for ofloxacin which was one of the
antibiotics rarely used in both hospitals. Results obtained show the important public health
hazard associated with the discharge of untreated hospital wastewater into the environment.
KEY WORDS: antibiotics, bacterial isolates, resistance, hospital
Path, 2001). The occurrence of strongly A myriad of substances such as selective environments such as hospitals, antibiotics, synthetic drugs, radionuclide, promotes, not only the growth of resistant and solvents, etc. are used in hospitals for bacteria, but also leads to an increase in the frequency of resistance bacterial genes and genetic elements such as plasmids.
application, many non-metabolized drugs Hospital wastewater, even if it is are excreted by the patients and enter the treated, may contain pathogenic drug- resistant bacteria, which constitute the wastewater may be present at levels that most dangerous single risk factor for could not only alter the ecology of the dissemination of pathogenic and drug- environment but also give rise to antibiotic resistant organisms in the environment resistance (Raloof, 1998).
(Diab et al., 2008). These resistant Acquired resistance to antibiotics bacterial species may be transmitted to may arise by cellular mutation or by acquisition of genetic elements in the form infection that cannot be treated by of plasmids or transposons (Russel and conventional antibiotics (Khachatourians, Akubuenyi et al., 2011 Nig. J. Microbiol. Vol. 25: 2243 - 2250 1998). Hence, the hospital wastewater with Isolation
its high content of multidrug resistant heterotrophic bacteria (TCHB).
bacteria and the presence of enteric Isolates of TCHB were obtained pathogens could pose a grave problem for through 10-fold serial dilution of the the community receiving such wastewaters (Cabrera et al. 2004; Chitris, 2004; physiological saline and pour plating of Somwang et al., 2005).
0.1ml aliquots in duplicates on Nutrient Agar (Difco Lab). Discrete colonies that untreated wastes of hospital and urban developed after incubation at 30oC for 48h sewage systems are released into rivers, were subcultured to obtain pure cultures lakes and other surface waters which also which were stored at 40C and used serve as sources of drinking water for local communities and poor homes and could characterization and biochemical analyses.
also help sources of water for the water Isolation of enteric bacteria
treatment plants that provide drinking One ml aliquots of appropriately water for the cities. Despite this practice, diluted samples were plated out (pour characterizations plate) in duplicates on MacConkey agar to wastewaters have been carried out in screen for coliform bacteria. To screen for Nigeria to determine the antibiogram of Salmonella spp. and Shigella spp., the the microbial diversity inherent in such samples were enriched in Selenite F broth discharges. The aim of this research for 8h and then pour plated on Salmonella- therefore was to determine the antibiotic Shigella agar and incubated for 24-48h. The samples were also enriched on wastewaters coming out of hospital drains alkaline peptone water (pH 8.3) and then in Calabar. The study was carried out on plated out (pour plate) on thioglycholate the wastewaters of the University of citrate bile salt sucrose (TCBS) agar, to Calabar Teaching Hospital (UCTH), and isolate Vibrio species. Pure cultures of the General Hospital Calabar (GHC). Both isolates were stored on nutrient agar slants hospitals are public health centers located characterization in Calabar, Cross River State, Nigeria, that identification (Krieg and Holt, 1994).
provide hospital services in a broad Characterization and identification of
category of illnesses and injuries.
The isolates were characterized and Materials and methods
procedures as described in Bergey s Collection of samples
Manual of Systemic Bacteriology (Krieg Wastewater samples were collected and Holt, 1994).
from UCTH and GHC both located in Antibiotic resistance studies
Calabar Metropolis, Nigeria. The sampling The standardized disc diffusion was performed according to the protocol method (Bauer et al., 1999) and the zone- of Nunez and Moretton (2007). Samples size interpretation chart of Kirby-bauer were collected over a 10h period taking (1966) was used for the in vitro samples every 2h for 16 days using sterile determination of the bacterial sensitivity to 500ml sample bottles. The same volume the various antibiotics selected, selection (250ml) of each partial sample was mixed of antibiotics was based on their usage in at the end of the day to obtain the the hospitals. The antibiotics used were; composite sample used for microbiological analyses. A total of 76 samples were collected from both hospital and all chloramphenicol (30g/ml), erythromycin samples were preserved in an icebox until transported to the laboratory.
Akubuenyi et al., 2011 Nig. J. Microbiol. Vol. 25: 2243 - 2250 (10g/ml), and cotrimoxazole (25g/ml). samples were 7.80•3.6 x 106 and 9.50•4.5 x 106 CFU/ml for TCHB, and 3.30•2.1 x (25g/ml), and pefloxacin (5g/ml) which 106 and 4.20•2.9 x 106 CFU/ml for are rarely used antibiotics in both hospitals coliforms respectively (Table 1). The were also included in this study. percentage of coliforms to total bacteria is Commercially prepared paper discs 42.31% and 44.21% respectively. A total impregnated with the various antibiotics of 125 isolates comprising of 65 from were mounted on Muller-Hinton agar UCTH and 60 from GHC were identified plates (Lab M) containing pure cultures of based on their colonial morphology and the various isolates. Each plate was their biochemical characteristics. They belonged to the following bacterial genera: suspensions containing 107 to 108 cells obtained using basic 0.5 Mcfarland solution (NCCLS, 1990). A total of 125 Bacillus; Staphylococcus and Proteus. All randomly selected isolates were tested and these bacterial genera except Proteus were results obtained were classified as resistant isolated from both hospital wastewaters. or sensitive. Inhibition zone diameters Proteus was isolated from only UCTH were measured after 18-24h of incubation.
wastewater samples. Tables 2 and 3 show the frequency of isolation of these counts from UCTH and GHC wastewater Table 1: Total bacterial and coliform counts of UCTH and GHC wastewaters
*Composite mean value of triplicate counts. Table 2: Frequency of isolation of the different bacterial genera from the wastewaters
of UCTH.

Total number of No. positive Frequency Staphylococcus Akubuenyi et al., 2011 Nig. J. Microbiol. Vol. 25: 2243 - 2250 Table 3: Frequency of isolation of the different bacterial genera from the wastewater of

No. of positive Frequency Staphylococcus Resistance patterns of isolates from (Table 4). Only one genus (Salmonella) both hospital wastewaters showed that all isolated from the GHC samples showed resistance to all the tested antibiotic resistance (MAR). Five genera antibiotics (Table 4). None of the bacteria (Salmonella, Klebsiella, Bacillus, isolated from the wastewater samples of Staphylococcus and Proteus) from the antibiotic resistance to all the antibiotics resistance to all the antibiotics tested Table 4: Antibacterial susceptibility profile of bacteria isolated from UCTH wastewater
Antibiotics tested % Resistance of single Akubuenyi et al., 2011 Nig. J. Microbiol. Vol. 25: 2243 - 2250 Table 4: Antibacterial susceptibility profile of bacteria isolated from
Antibiotics tested s. igella. lebsiella % Resistance of single All the isolates from the UCTH only Salmonella had developed resistance wastewater samples showed resistant to all to all twelve antibiotics used in this study the antibiotics (amoxycillin, gentamycin, (Table 5). It was also found that all the chloramphenicol, bacterial isolates from the GHC had erythromycin, tetracycline, streptomycin developed resistance to some of the and cotrimaxazole) commonly used in the (chloramphenicol, hospital except Ciprofloxacin. Escherichia erythromycin, streptomycin, tetracycline coli and Pseudomonas from UCTH and and cotrimaxazole) alone from GHC were hospital. Only Salmonella and Staphyloc sensitive to Ciprofloxacin (Tables 4 & 5). More organisms - Shigella, Escherichia ofloxacin which was also rarely used in and Streptococcus showed sensitivity to that hospital.
antibiotics). Shigella and Escherichia were The TCHB and coliform counts in sensitive to ceftriazone, another not investigated were similar to counts Streptococcus in UCTH and Streptococcus and Escherichia Argentina by Nunez and Moretton (2007). sensitivity to Pefloxacin – third rarely used Similar results were also obtained for the antibiotic employed. Data obtained from the GHC wastewater analyses showed that wastewater, Brazil (Ortolan, 1999) and the Akubuenyi et al., 2011 Nig. J. Microbiol. Vol. 25: 2243 - 2250 University of Ilorin Teaching Hospital ability/predisposition to develop resistance wastewater (Olayemi and Opaleye, 1990). under conditions of antibiotic selective The counts obtained for the bacterial pressure (e.g. hospital environments) groups at the two studied sites indicated might be responsible for the acquisition of high pollution level. The safe discharge the antibiotic resistance capacity of these level of such wastewaters should not bacteria. The spectrum of resistance exceed 100CFU/ml for TCHB and 30CFU/ml for coliforms (EPA Guidelines, Escherichia indicates that the multidrug 2003). Out of the 7.80•3.6 x 106 CFU/ml resistance genes responsible for the unique total bacteria count obtained in the UCTH characteristics observed might also be domiciled in some of these bacterial coliforms while 44.21% of the 9.50•4.5 x CFU/ml of total bacteria count obtained from GHC were coliforms (Table resistance among the bacterial isolates was 1). Escherichia coli and Streptococcus spp shown for the most commonly used were the most frequently isolated bacteria antibiotics in both hospitals (Tables 3 & (Table 2). All the isolates had multiple 4). This result thus lays credence to a antibiotic resistance, with most of the previous report by Islam et al. (2008) isolates from both hospital samples which claims that resistance development (Tables 3 & 4) exhibiting resistance to at was directly related to the use of least 6 antibiotics. Resistance to all of 9 commonly used antibiotics was recorded It is widely acknowledged that the for 7 genera in UCTH namely: Salmonella, selection and dissemination of antibiotic Shigella, Streptococcus, resistant bacteria in nature should be Staphylococcus and Proteus order to maintain the (Table 4), whereas 4 genera (Salmonella, ecological balance that favours the Shigella, Klebsiella and Streptococcus) predominance of a susceptible bacterial showed resistance to the same nine flora in the environment and to ensure antibiotics in GHC (Table 5). Previous effective treatment against infectious reports have implicated E. coli strains as diseases in humans and farm animals. the main carrier of antimicrobial resistance Thus, the indiscriminate use of antibiotics in faecal flora, as resistance in other and the discharge of untreated antibiotic- species was rare especially in the absence of antimicrobial selection (Osterblad, et al. environment may disrupt the microbial 2000; Guardaasi, et al., 1998). Results balance in nature in favour of resistant obtained in this study however have shown that other genera can acquire multiple important public health hazard that may be antibiotic resistance capacity and could surpass E. coli in exhibiting resistance to discharge of hospital effluent into the environment without adequate treatment.
The authors wish to thank the laboratory crew of the Department of Bauer, A. W., Kirby, W. M. M., Strerris, J. C. and M. Turk, (1999). Antibiotic University of Technology, Nigeria for susceptibility testing by a standard their technical assistance.
single disk method. American Journal of Clinical Pathology. 45:493-496.
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Systematic Bacteriology. William and Wilkins, Baltimore Ltd.
Guardaassi, L., Petersen, A., Olsen, J. and Lamikanra, A. and I. N. Okeke, (1997). A A. Dalsgaard, (1998). Antibiotic resistance in Acinetobacter spp. urban/rural divide on the incidence isolated from sewers receiving of antibiotic resistance in E. coli. waste effluents from a hospital and Biomedical Letters. 55:91-97 a pharmaceutical plant. Appl. Environ Microbial. 61:3499-3502.
Leprat, P. I., (1998). Les rejets liquides hospitaliers, quels agents et quelles Islam, M. J., Uddin, M. S. Hakim, M. A. Das, K. K. and M. N. Hassan, Techniques hospitalieres. 632:49- (2008). Role of untreated liquid hospital waste to the development of antibiotic resistant bacteria. J. Lery, S. B., (2002). How misuse of Innov. Dev. Strategy. 2 (2):17-21.
antibiotics destroy their curative powers. The antibiotic paradox. Jeannette M. A., Lang, K. S, Lapara, T. M. Oxford University Press. 39-41.
Gonzatez, G. and R. S. Singer, (2007). Evaluating the effect of Nunez, L. and J. Moretton, (2007). chlortetracycline Disinfectant-resistant bacteria in proliferation of antibiotic-resistant bacteria in a stimulated river water wastewater. Braz. J. Microbiol. 38
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Okello, D., Konde-Lule, J., Lubarga, R. and J. Arube-Wani, (1997). Waste disposal in private medical clinics Agricultural use of antibiotics and in Kampala, Uganda. J. Clin. the evolution and transfer of antibiotic-resistant bacteria. CMAJ. 159(9):1129-1136.
Olayele, A.B. and F.I. Opaleye, (1990). Kummerer, K. and A. Henninger, (2003). coliform bacteria isolated from hospital and urban wastewater. Biotechnology. 6. 285-288. Clin Microbiol Infect. Ortolan, M. G. S., (1999). Avaliacao do effluente do hospital de Clinicas de Kummerer, K., (2001). Drugs in the environment: emission of drugs, diagnostic aids and disinfectants into waste-water by hospitals in relation to other sources. A Review antibioticos. Porto Alegre, 115.
Osterblad, M., Hakanen, A., Manninen, R., Akubuenyi et al., 2011 Nig. J. Microbiol. Vol. 25: 2243 - 2250 Meurman, O., Huovinen, P. and P. Kotilainen (2000). A between– Lancet Infect. Dis 3:794-803.
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