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2010518_antibiotic_resistance_faecal_bacteria

Atieno et al / Journal of Biology (2013), Vol. 01, Issue 05, pp. 106-111 Research Paper
Antibiotic Resistance of Faecal Bacteria Indicators and
Pathogens Isolated from Sludge and Wastewaters of
Abattoirs in Nairobi, Kenya
Nyamboya Rosemary Atieno1, Okemo Paul Owuor1* and Ombori Omwoyo1 1Department of Plant and Microbial Sciences, Kenyatta University, P.O. Box 43844-00100, Nairobi, Kenya *Tel: +254-722942072 *E-Mail: [email protected]., [email protected].
Abstract

Bacterial antibiotic resistance has become a serious problem among pathogenic bacteria and has led to increased concern
surrounding environmental risks and potential spread of resistance in microbial species. Besides having clinical consequen-
ces, resistant bacteria of animal origin may be the source of determinants of resistance for the possible transfer to human
strains. The objectives of the present study were to determine the abundance and distribution of antibiotic and multiple drug
resistance among faecal bacteria indicators and pathogens found in wastewaters of animal abattoirs in Nairobi, Kenya.
Standard microbiological methods were used to isolate and identify faecal streptococci, faecal coliforms, Vibrio and
Salmonella species. Sensitivity to antibiotics was determined by agar diffusion method. The mean intermediary sensitive
case (8.1% (±5.6)) was significantly lower (p=0.00) compared to the mean sensitive (41.3% (±23.1)) and mean resistant
(50.6% (±22.3)) cases at p<0.05. Isolates showed high resistance to lincomycin (90%), ampicillin (80%), and methicillin
(72.5%) and low resistance to chloramphenicol (22.5%). The results provided dynamics of resistance development in warm
blooded animals usually consumed by humans. Multiple antibiotic resistance index was >0.2 indicating high risk of exposu-
re to the various antibiotics.
Keywords: Antibiotic Resistance, Bacterial Indicators, Pathogens, Sewage and Abattoirs
1. Introduction
During the past sixty years, antimicrobials were extensive-
warned that misuse of drugs is making them ineffective as ly used as growth promoters in breeding practices as well treatments for various diseases and ailments. Antibiotics as in human and veterinary medicine. Nevertheless concer- are easily available over the counter with buyers not havi- ns about the use and disposal of these pharmaceuticals ha- ng to submit a doctor's prescription. This easy availability ve been rising during the past decade (Garcia-Armisen et means many people are either overdosing or under dosing al, 2011) because of their impact on both human health and or simply buying antibiotics when they don't need them. the environment. Bacterial resistance to antibiotics has be- Resistance is typically common where antibiotics are heav- come a serious problem among pathogenic bacteria, wh- ily used (hospitals, long term care centres and large livest- ich has led to an increased concern surrounding environm- ock operations), although antibiotic resistant bacteria are ental risks and potential spread of antibiotic resistance am- also shown to be present in wastewater, surface water, gro- ong microorganisms. In Kenya, a bacterium, Neisseria go- und water, sediments and soils and increasingly in surface norrhea, which was previously sensitive to penicillin, has aquatic environments (Zbigniew, 2005; Baquero et al, now developed resistance to this drug (John, 2012). Senior 2008 and Zhang et al, 2009). The rapid increase in multip- ministry of health officials and doctors in Kenya have le antibiotic resistant in aquatic bacteria is partially due to Available online at www.scientific-journals.co.uk Atieno et al / Journal of Biology (2013), Vol. 01, Issue 05, pp. 106-111 the ability of those bacteria to transfer antibiotic resistance 1.3. Antibiotic Sensitivity Test
markers among the bacterial population by cell to cell con- tact. The prolonged exposure to low doses of antibiotics le- Sensitivity to antibiotics was determined by the agar diffu- ads to the selective proliferation of resistant bacteria, whi- sion technique recommended by the NCCLS (National ch could horizontally transfer genes to other bacterial spec- Committee for Clinical Laboratory Standards) (NCCLS, ies (Aminov & Mackie, 2007 and Zbigniew et al, 2010). 2003) on Mueller-Hinton agar (Oxoid) using the following antibiotic impregnated disks: ampicillin (25 µg); cotrimox- In the livestock sector, different types of farm animals are azole (25 µg); streptomycin (10 µg); chloramphenicol (30 capable of carrying a wide range of zoonotic pathogens µg); kanamycin (30 µg); gentamicin (10 µg); penicillin G (Swai & Schoonman, 2012). Livestock often act as non- (1 unit); methicillin (5 µg); minocycline (30 µg); lincomy- symptomatic carriers of human pathogens such as E. coli cin (2 µg); erythromycin (15 µg); tetracycline (25 µg) and 0157, Salmonella species and Campylobacter, which are sulfamethoxazole (200 µg). Interpretation of the results na- rarely detected during routine ante-mortem examination mely sensitive (S), Intermediary Resistant (IR) and resista- and their wastes may contain high concentrations of the nt (R) was made in accordance to the standard measureme- organisms. Animal waste can therefore contaminate human nt of inhibitory zones in millimetre (mm). Multiple Drug and animal drinking water sources and even soil when us- Resistance (MDR) index values were calculated using the ed as manure (Christina et al, 2012). formula by Lee et al (2009) as follows: In relation to the health of animals and food safety concern for human consumption it is crucial to control the suscepti- bility of bacteria in animal wastewaters to antibiotics. The X=Total of antibiotic resistant case contribution of abattoirs and associated wastewaters is Y=Total of antibiotic used in the study rarely considered and yet abattoirs are potential sources of Z=Total number of isolates enteric bacteria that could possess antibiotic resistance ge- 1.4. Data Analysis
nes. Due to the limited scientific documentation regarding these aspects, this study was conducted to evaluate the pre-
SPSS computer software version 16.0 was used for data
valence of Antibiotic Resistance (AR) and Multiple Drug entry and statistical analysis. Groups significance tests we- Resistance (MDR) among two groups of faecal pollution re performed using one way ANOVA at 5% significance indicators; Faecal Streptococci (FS) and faecal coliforms and two pathogenic bacteria i.e. Vibrio and salmonella level and P value of <0.05 was considered as significant. The means were separated using Tukey's Honest Signific- ance Difference (HSD) test at 5% level. 2. Materials and Methods
3. Results
1.1. Sample Collection and Preparation
3.1. Antibiotic Resistance Patterns
The wastewater and sludge samples were collected from High percentages of bacterial isolates were resistant to cattle abattoir in Kayole, sheep and goat abattoir in Kiama- lincomycin (90.0%), ampicillin (80.0%) and methicillin iko, Nairobi, Kenya. The samples were collected in clean (72.5%) (Table 1). Some isolates were sensitive to gentam- sterile 200 mL plastic bottles and transported to Kenyatta icin (72.5%) and chloramphenicol (75%), hence the latter University laboratory in an ice cooler box for analysis. two were considered to be the most effective antibiotics. Wastewater samples that were not analyzed within four The study shows that in Kenya the drugs of choice for trea- hours were stored at a temperature of 4 °C. All samples tment of the isolates are chloramphenicol (75.0%), genta- were analyzed within 24 h. micin (72.5%) and minocycline (60.0%). Lincomycin (5.0 %), ampicillin (12.5%), tetracycline (22.5%) and sulfamet- 1.2. Isolation and Identification of Bacterial Isolates
hoxazole (25%) are ineffective according to the study. The mean intermediary sensitive case {8.1% (±5.6)} was signi- Standard microbiological methods (Mariita & Okemo, ficantly lower (p=0.00) compared to the mean sensitive 2009) were used to isolate Faecal Coliforms (FC), Faecal {41.3% (±23.1)} and mean resistant {50.6% (±22.3)} cas- Streptococci (FS), Vibrio and Salmonella species in the sa- es at p<0.05. mples of wastewaters and sludge. Pigmentation of the col- onies and Gram's staining followed by standard biochemi- When sources of isolates are considered (Table 2), a high cal characterization {such as mortility, urease, TSI (triose percentage of cattle wastewater isolates showed resistance sugar iron), glucose fermentation, indole, citrate utilizati- to ampicillin (100%), lincomycin and tetracycline (87.5%) on, and the cytochrome oxidase tests} were used to confi- and methicillin (75%). None of the cattle wastewater isola- rm the bacterial isolates. tes was resistant to minocycline. Available online at www.scientific-journals.co.uk Atieno et al / Journal of Biology (2013), Vol. 01, Issue 05, pp. 106-111 Table 1. Antibiotic Sensitivity of Randomly Selected Bacterial Isolat-
omycin and ampicillin (100%) and penicillin (75.0%) and es (n=40) to 13 Types of Antibiotics
only 12.5% were resistant to chloramphenicol. Bacter-ial isolates from goat and sheep sludge mostly showed resista- Resistance
Sensitive
nce to lincomycin (100 %), ampicillin (87.5%) and none of Antibiotic (ng)
Sensitive
the isolates was resistant to chloramphenicol. High perce- ntages of the sheep wastewater isolates were resistant to lincomycin (87.5 %) but none of the isolates was resistant Chloramphenicol (30) to cotrimoxazole. Goat wastewater isolates were mostly resistant to methicillin (100.0%), lincomycin and tetracycl- Cotrimoxazole (25) ine (75%) while only 12.5% were resistant to kanamycin Erythromycin (15) and gentamicin. There was no significant difference (p= 0.971) in resistance to antibiotics of bacterial isolates coll- ected across the waste-waters and sludge samples. All bacteria isolated from cattle sludge were resistant to lincosamides (100%) (Table 3). Overall, a high percentage Minocycline (30) of bacterial strains isolated from all sites were more resist- Penicillin G (1 unit) ant to lincosamides, and β-lactam classes of antibiotics co-mpared to aminoglycosides, macrolides, sulfonamides, ph- Streptomycin (10) enolics and tetracyclines. Only 22.5% (±18.5) of the studi- Sulfamethoxazole ed isolates showed resistance to phenicols. There was sign- ificant difference (p=0.00) in resistance to β-lactams, linc- Tetracycline (25) osamides and phenicols at p<0.05. 8.1b (±5.6) 41.3a (±23.1) 3.2. Multiple Antibiotic Resistance
n= total number of isolates tested; Inter.= Intermediary Means having the same letters within the row are not significantly different according to Tukey's HSD at 5% level All the studied bacterial strains exhibited resistance to mo-
Table 2. Resistance of Bacterial Isolates Collected From Wastewaters and Sludge
Percentage of AR Among Isolates in Wastewaters and Sludge
Antibiotic (ng)
Chloramphenicol (30) Cotrimoxazole (25) Erythromycin (15) Minocycline (30) Penicillin G (1 unit) Streptomycin (10) Sulfamethoxazole (200) Tetracycline (25) AR-antibiotic resistance, 1-cattle wastewater, 2-cattle sludge, 3-goat wastewater, 4-sheep wastewater and 5-goat and sheep sludge. Means followed by the same letters within the row are not significantly different according to Tukey's HSD at 5% level. Most cattle sludge isolatees also showed resistance to linc- re than 3 antibiotics (Table 4). However, the patterns of Available online at www.scientific-journals.co.uk Atieno et al / Journal of Biology (2013), Vol. 01, Issue 05, pp. 106-111 resistance among the 40 cultures varied as represented in ance genes which are exchanged by bacteria from different Table 4. Of all the studied bacterial isolates, 52.5% (21) ecosystems (Dang et al, 2008). The five sources of bacteri- showed 6 Multiple Antibiotic Resistance (MAR) pattern al contamination characterized in this study were investig- (i.e. resistance to 6 of the 13 antibiotics tested). Only a sm- ated because they differ in terms of the origin of the bacte- all percentage of the isolates (2.5%) showed a 4 MAR patt- ria strain they release in the environment (cattle, goat and ern. None of the studied bacteria showed a 0-3 and 9-13 sheep) and in the expected exposure of these bacteria to antimicrobial selective pressure.
Table 3. Resistance of Bacteria to Antibiotics With Respect to the Nature of the Antibiotics (in %)

Wastewaters and Sludge
Antibiotic
Mean (±SD)
AM-aminoglycosides (kanamycin, gentamicin, streptomycin), SUL-sulfonamides (sulfamethoxazole, cotrimoxazole), LA-β-lactams (ampicillin, penicill-in, methicillin), LI-lincosamides (lincomycin), MA-macrolides (erythromycin), PH-phenicols (chloramphenicol) and TET-tetracycline (tetracycline, mi-nocycline). 1-cattle wastewater, 2-cattle sludge, 3-goat wastewater, 4-sheep wastewater and 5-mixture of goat and sheep sludge. Means followed with same letters within the column are not significantly different according to Tukey's HSD at 5% level Table 4. Patterns of MDR of Faecal Coliforms, Faecal Streptococci
Table 5. Multiple Antibiotic Resistance (MAR) Value of Bacterial
and Vibrio and Salmonella Species (n=40) to 13 Antibiotics
Isolates
Types of Antibiotics
Bacterial Isolate
MDR Value
Antibiotics
of Strains
Tet, sulf, pen, ery, kan, gen,cot, chlo Salmonella species Linc, amp, met, tet, ery, sulf, mino Linc, amp, met, tet, ery, pen, gen Faecal streptococci Linc, amp, kan, met, sulf, pen Vibrio species Linc, amp, kan, tet, strep, cot Linc, sulf, strep, cot, mino, chlo, Bacterial isolates were most resistant to lincomycin, ampi- Amp, met, ery, chlo, strep, cot cillin and methicillin and most sensitive to chloramphenic- Tet, kan, gen, strep al, gentamicin and cotrimoxazole. These results, therefore, show that ampicillin, lincomycin and methicillin are not Amp:ampicillin, Linc:lincomycin, Pen:penicillin, Met:methicillin, Ery: erythromycin, Tet:tetracycline, Cot:cotrimoxazole, Strep:streptomycin, effective in the control of these bacteria since high percent- Kan:kanamycin, Gen:gentamicin, Sulf:sulfamethoxazole, Chlo-chloram- ages of the isolates (i.e. 80%, 90% and 72.5%, respective- phenicol and Mino:minocycline ly) were resistant to these antibiotics. Bacterial resistance to erythromycin, streptomycin, penicillin, ampicillin, amo- All MAR index values were found to be more than 0.20, xicillin, kanamycin, tetracycline, oxytetracycline and chlo- where the highest MAR index was exhibited by FC (0.40), ramphenicol has been reported (Roberts, 2011). followed by Salmonella spp. (0.35), Faecal Streptococci (0.30) and Vibrio spp. (0.225) (Table 5). The present study demonstrated that bacterial isolates were resistant to antibiotics commonly used as feed additives 3. Discussion
(tetracycline, streptomycin and sulfonamides) or therapeut- ics (penicillin and tetracycline). According to Zbigniew et Bacterial resistance to antibiotics is a growing medical and al (2005) bacteria resistance to antibiotics depends on the ecological problem worldwide generated by the selection chemical structure of the antibiotics; this is confirmed by process following the indiscriminate use of antibiotics results of the present study. (Meirelles-Pereira et al, 2002). Environmental bacteria ma- y play an important role as reservoirs of antibiotics; resist- All the bacteria isolated from cattle sludge were resistant Available online at www.scientific-journals.co.uk Atieno et al / Journal of Biology (2013), Vol. 01, Issue 05, pp. 106-111 to lincosamide antibiotics and therefore studied bacteria of Veterinary Services of Kenya is also acknowledged for are capable of detoxifying these antimicrobial agents. Lin- giving us ethical clearance. cosamides are one of the commonly used antibiotic classes in human and veterinary clinical practice (Andreozzi et al, References
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