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Journal of sciences multi-antibiotic resistance of some gram negative bacterial isolates from poultry litters of selected farms in benue state

Volume 2 No.8, August 2012 ISSN 2224-3577
International Journal of Science and Technology
2012 IJST. All rights reserved Multi-Antibiotic Resistance of Some Gram Negative Bacterial Isolates from
Poultry Litters of Selected Farms in Benue State
1 Hemen , J.T., 2Johnson, J.T., 3Ambo, E.E., 4Ekam, V.S, 5Odey, M.O., 4Fila, W.A.
1Department of Biological Sciences, College of Natural Sciences University of Mkar, Mkar, P.M.B. 017. Benue State Nigeria. 2Department of Biochemistry, College of Natural Sciences University of Mkar, Mkar, P.M.B. 017. Benue State Nigeria. 3Department of Microbiology, faculty of Sciences University of Calabar, P.M.B 1115 Calabar, Cross River State Nigeria. 4Department of Biochemistry, College of Medical Sciences University of Calabar, P.M.B 1115 Calabar, Cross River State Nigeria. 5National Research Institute for Chemical Technology P.M.B. 1052 Zaria, Kaduna State Nigeria. ABSTRACT
This study was aimed at isolating and identifying Shigella, Salmonella and Escherichia coli bacteria associated with poultry litter
obtained from the University of Mkar Community, Adekaa, Gboko central and GRA Gboko. The study also determines the antibiotic
sensitivity patterns of the associated bacteria. The bacteria were isolated and identified phenotipically following standard microbiological
methods. The antibiotic sensitivity patterns of the isolated bacteria tested against Septrin, Chloranphenicol, Sparfloxacin, Ciprofloxacin,
Amoxacillin, Augmentin, Gentamycin, Pefloxacin, Tarivid and Streptomycin. Shigella and Salmonella were completely resistant to
chloranphenicol, augmentin, pefloxacin, amoxicillin. Shigella were also resistant to all the antibiotics excepts Septrin and ciprofloxacin
showed they are intermediate to the drugs. Percentage antibiotics susceptibility pattern of gram negative bacteria isolated from poultry
litter showed all bacterial isolates (100%) were resistant to Chloranphenicol while most of the isolates were susceptible to Amoxacillin.
All the bacterial isolates showed high level (10.2 MAR index) antibiotic resistance. The study found that there was close (R= .516)
association between the isolates and antibiotics. There was also significant relationship (X2= 5.871) between isolates and occurrence at
different sample sites. The study therefore recommends proper information dissemination to farmers and poultry feeds producers on
dangers of antibiotic resistant strains, use of Probiotics in animal feeds to prevent gastro-intestinal infections and use of regulations to
control poultry litter disposal.

Keywords: Multi-Antibiotics, Resistance, Gram Negative, Bacterial Isolates, Poultry Litter.
1. INTRODUCTION
today. In intensively reared food animals, antibiotics are administered for therapeutic purpose and as Antimicrobial growth promoters (AMGPs) to the whole flock rather than individuals Poultry is a major fast growing source of meat in the [6]. Resistance to antibiotics can either be naturally occurring for world today, representing a quarter of all the meat a particular organism/drug combination or acquired resistance, produced in 2000 [1]. The modern poultry industry can where mis-use of anti-microbials results in a population being produce market ready broiler chickens in less than six weeks. exposed to an environment in which organisms that have genes This accomplishment is done through genetic selection, improved conferring resistance (either spontaneously mutated or through keen health management practices involving DNA transfer from other resistant cells) have been able to usage of antibiotics as therapeutic agents to treat bacterial flourish and spread. Hence, the antibiotic selection pressure for diseases in intensive farming systems [2]. Acquired resistance resistance in bacteria in poultry is high and consequently their against frequently used antibiotics have been observed since the fecal flora contains a relatively high proportion of resistant introduction of these antimicrobial agents in human and bacteria [6]. Resistant strains from the poultry gut readily soil veterinary medicine [3]. The use of antibiotics is a major factor in poultry carcasses and when consumed, they alter or affect human emergence, selection and dissemination of antibiotic resistant endogenous flora [7]. Gene transfer occurs majorly in vivo microorganisms in both veterinary and human medicine [4]. The between gastrointestinal tract bacteria and as rise in antibiotics resistance have been reported in the past two identical resistant genes are present in diverse bacterial species decade [5] and antibiotic resistance still remains a global problem from different hosts [8]. Volume 2 No.8, August 2012 ISSN 2224-3577
International Journal of Science and Technology
2012 IJST. All rights reserved In light of this, there is probability that mos Gram staining
that threaten human health may soon be resistant to all known antibiotics [9]. Certain antibiotics however are critical to Gram staining was carried out as described by [19] to identify the human infections caused by multidrug resistant pathogens, or gram negative bacteria. because alternative therapies are less effective or are associated with side effects [10]. The determination of the effectiveness of Oxidase Test
antimicrobial agents against specific pathogens-either human or animal source- is essential for proper therapy [11]. The Oxidase test was performed as described by [15]. development of resistance to antimicrobial drugs is a serious problem worldwide, which threatens the ability to treat infections Antibiotics Disk
in animals and humans [1]. Most resistance problem probably arose from inappropriate use of antibiotics which exposes The bacterial isolates were tested for resistance to 10 antibiotics infectious agents to sub therapeutic doses of antimicrobial agents produced by MAXIDISC (Maxi care medical Laboratories Ltd., [12]. Moreso, the use of antimicrobials in agriculture especially Nigeria). These were: Septrin (30 μg), Chloranphenicol (30 μg), as growth promoters, chemotherapeutic and prophylactic agents Sparfloxacin (10 μg), Ceprofloxacin (10 μg), Amoxacillin (10 in food animals bacteria are of public health implication [13, 14]. μg), Augmentin (30 μg), Gentamycin (10 μg), Pefloxacin Thus, it became imperative to provide information on the streptomycin (30 μg), Tarivid (10 μg) and Streptomycin (30μg). multiple antibiotic resistant on some Gram-negative bacteria This testing was performed using the standard disc diffusion isolated from poultry litter from selected farms in Gboko method [21]. The antibiotics susceptibility pattern of the isolates was interpreted using Progressive Diagnostics Manufacturers (PDM) Interpretative Chart. 2. MATERIALS AND METHODS

Antibiotic Sensitivity Testing

Sample Collection
The test was carried out as described by [11]. Four hundred and eighty (480) samples from GRA Gboko, Central Gboko, Adekaa and University of Mkar Measurement of Zone of Inhibition
community settlement area were freshly collected from the month of June to August 2011, at time interval of 3-4 hours between 8: The metric ruler was placed across zones of inhibition, at the am and 5: pm in sterile polypropylene containers. That was widest diameter, and measured from one edge of the zone to morning afternoon and evening and transported to the laboratory the other edge in accordance to [12]. Zone diametesr were within 30 minutes for analysis. reported in millimeters, as ‘S' (sensitive), ‘R' (resistant), or ‘I' (intermediate) as describe by [12]. Isolation and Identification of Bacteria

Sensitivity
Isolation and identification of bacteria were done based on their morphological, Gram staining, cultural, and biochemical A clear, ‘'halo'' (technically known as a ‘'plaque or zone of properties described by Chessbrough, [15]. representatives of the inhibition) that appeared around the antibiotic disk, indicating an different colonies were selected according to their morphological absence of bacteria as reported by [12]. characteristics and purified by successive sub culturing on Salmonella-Shigella agar and identified phenotipically based on standard methods [16, 17]. Salmonella- Shigella and Mackonkey Agar plate were used to differentiate among Gram-negative bacteria. E. coli Somewhat cloudy plaque appearance indicates not all the bacteria bacteria ferment the lactose in the media; resulting in bacterial in the area around the disk were killed i.e using the method growth with a pink color (they do not produced any hydrogen discuss by [22]. sulfide). Member of the genus Salmonella does not ferment lactose, but do produce hydrogen sulfide gas, so the resulting Resistant
colonies were colorless as describe by [18]. In this case, the antibiotic disk would have no discernable plaque Volume 2 No.8, August 2012 ISSN 2224-3577
International Journal of Science and Technology
2012 IJST. All rights reserved Stastual Analysis
causes discovery of more resistant bacteria. This the bacteria associated with poultry litter in this study. Thus, the Regression analysis was used to estimate overall sensitivity and antibiotic selection pressure for resistance by bacteria in poultry resistant pattern similarities of the bacterial resistance using their is high and as a result their fecal floor contains higher proportion zones of inhibition[1]. Chi-square was also employed to test the of resistant bacteria [6]. The Multiple Antibiotic Resistance level of significance between the isolates and the sample sites (MAR) index is defined as a/b where ‘a' represents the using Predictive Arithmetic Software (PASW). er of antibiotics to which the particular isolate is resistant and ‘b' the number of antibiotics to which the isolate is exposed [28]. MAR index values higher than 0.2 were considered to have 3. RESULTS
originated from high- risk sources where antibiotics are often used. MAR index values of less than or equal Table1 shows antibiotic susceptibility pattern of some indicates strain originated from sources where antibiotics are Gram negative bacteria isolated from poultry litter. The result seldom or never use. Statistical evaluation estimate overall showed that E. coli had the highest rate of resistance with 0.8 similarities of the bacterial resistance using their zones of mar index, followed by Salmonella (0.6) and Shigella (0.3). inhibition [1]. Conclusively, the present results provide evidence Moreso, Shigella had highest number of intermediate with five that poultry litter can serve as an environmental reservoir of (5), while E. coli and Salmonella two (2) respectively. multiple antibiotics resistant bacteria and hence as potential route Furthermore, Salmonella was susceptible to (CPX and AM) while for the entry of multidrug resistant zoonotic pathogens into E. coli had none susceptibility response as also shown in table 1. human population. This have very important implications for human health, as multidrug resistant infections are difficult to 4. DISCUSSION
treat and often requires expensive antibiotics and long term therapy. This can substantially increase the cost of treatment and The rise in antibiotics resistance had been reported in the past two decade[5] and antibiotic resistance still remains a global problem today. In intensively reared food animals, antibiotics are REFERENCES
administered for therapeutic purpose and as Antimicrobial growth promoters (AMGPs) to the whole flock rather than individuals [1] Adeleke E.O. and Omafuvbe B.O, (2011). Antibiotic [6]. Resistance to antibiotics can either be naturally occurring for Resistance of Aerobic Mesophilic Bacteria Isolated from a particular organism/drug combination or acquired resistance, Poultry Faeces. Research Journal of Microbiology, 6: where mis-use of anti-microbials results in a population being exposed to an environment in which organisms that have genes conferring resistance (either spontaneously mutated or through [2] Apata, D.F., 2009. Antibiotic resistance in poultry. Int. DNA transfer from other resistant cells) have been able to J. Poult. Sci., 8: 404-408. flourish and spread. High level of antibiotic resistance was observed in this study with Ciprofloxacin, Amoxacillin, [3] Smith, D.W., 1999. Decreased antimicrobial resistance Augmentin, Gentamycin, Pefloxacin, Tarivid and Streptomycin after changes in antibiotiuse. Pharmacotherapy, 19: 129- to mention but just few. From the four sampling sites about three genus of Gram negative bacteria were isolated; these include [4] Tollefson, L. and W.T. Flynn, (2002). Impact of Shigella, Salmonella and Escherichia coli. These organisms are antimicrobial resistance on regulatory policies in of public health importance [14]. They all showed multiple veterinary medicine: Status report. AAPS Pharmsci., 4: resistance to above mentioned antimicrobial agents. However, the high level of E. coli resistance to tested antibiotic seems to correspond with the report of [23]. This may be attributed to [5] Kapil, A., (2004). The challenge of antimicrobial constant contact between feed, poultry birds and fecal dropping resistance: Need to contemplate. Indian J. Med. Res., as also reported by [24]. Most of the isolated bacteria like E. coli 121: 83-91. and Salmonella showed high level of resistance more than Shigella, and they constitute normal flora of the intestinal tract of [6] Van der Bogaard, A.E. and E.E. Stobberingh, (1999). poultry as reported by [25]. The bacterial isolates showed high Antibiotic usage in animals: impact on bacterial level of antibiotic resistance against all used antibiotics. The resistance and public health. Drugs., 58: 589-607. result was in agreement with [26, 27] who reported that the abuse [7] Van der Bogaard, A.E. and E.E. Stobberingh, (2001). and misuse of antimicrobial agents for growth promotion and Recent trend in antibiotic usage and bacterial resistance. prevention of diseases has impressed a selective pressure that Drugs., 65: 102-110. Volume 2 No.8, August 2012 ISSN 2224-3577
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2012 IJST. All rights reserved [8] Scott, K.P., 2002. The role of conjugative transposons in [18] Isenberg, G.V. (1992). The Shorter Bergey's Manual of spreading antibiotic resistance between bacteria that inhabit the gastrointestinal tract. Cell Mol. Life Sci., 59: Wilkins Co., Baltimore, USA [19] Gaffa, T and Azoro, C (2005). Bacteriology for [9] Comparison of Biologist, Caterers and Food Technologists. Amana antimicrobial resistance among human and animal Printing and Advert limited kaduna.pp.47-52 enterococci with emphasis on the macroline- lincosamide-streptogramin group. Ph.D. thesis, ghent [20] Cheesbrough, M. (2004). Medical laboratory manual for University, Belgium tropical countries. Vol.IV. Microbiology. pp. 503-518. [10] Akond, M.A., S. Alam, S.M.R. Hasan, S.N. Uddin and [21] Clinical and Laboratory Standards Institute, 2006. M. Shirin, (2008). Antibiotic resistance of Vibrio Performance standards for antimicrobial susceptibility cholera from poultry sources of Dhaka, Bangladesh. testing; sixteenth informational supplement. M100-S16, Adv. Biol. Res., 2: 60-67. Vol. 26 No. 3, pp: 1-37. [11] Prescott, L.M., J.P. Harley and D.A. Klein, 2005. [22] Salehi, T.Z. and S.F. Bonab, (2006). Antibiotics Microbiology. 6th Edn., McGraw-Hill Co., New York, susceptibility pattern of Escheichia coli strains isolated from chickens with colisepticemia in Tabriz Province, Iran. Int. J. Poult. Sci., 5: 677-684. Antimicrobial resistance: an ecological perspective. [23] Adegunloye, D.V., (2006). Microorganisms associated Report of an American Academy of Microbiology with poultry faeces. J. Food Agric. Environ., 4: colloquium. July 16–18, 1999, San Juan, Puerto Rico. [13] Witte W (1998) Medical consequences of antibiotics use [24] Vellinga, H.A. and Van-loock, J.Q. (2002). Bacteriology in agriculture Science for Biologist, Caterers and Food Technologists. Amana Microbiol Biotechnol (2009) 25:713–719 719 Printing and Advert limited kaduna.pp.47-52 [14] Heuer H, Smalla, K. (2007). Manure and sulfadiazine [25] Esposito, S. and S. Leone, (2007). Antimicrobial synergistically increased bacterial antibiotic resistance in treatment for Intensive Care Unit (ICU) infections soil over at least two months. Environ Microbial 9:657– including the role of the infectious diseases specialist. Int. J. Antimicrob. Agents, 29: 494-500. [15] Cheesbrough, M. (1985). Medical laboratory manual for [26] Cloud, S.S., J.K. Rosenberger, P.A. Fries, R.A. Wilson tropical countries. Vol.II. Microbiology. pp. 400-480. and E.M. Odor, (1985). In vitro and in vivo characterization of avian Escherichia coli Serotypes, [16] Harrigan, W.F. and M.E. McCance, (1976). Laboratory metabolic activity and antibiotic sensitivity. Avian Dis., Methods in Food and Dairy Microbiology. 1st End. Academic Press, London, pp: 25-29. Coli 0157:H7 from apparently healthy animals. Afr J [27] Muhammad, M., L.U. Muhammad, A.G. Ambali and Biotechnol 2:322–324 A.U. Mani, (2010). A survey of early chick mortality on small-scale poultry farms in jos, central Nigeria. Int. J. [17] Buchannan, R.E. and N.E. Gibbons, (1985). Bergey Poult. Sci., 9: 446-449. Manual of Determinative Bacteriology. Vol. 1. 9th End. Williams and Wilkins, Baltimore, USA. [28] Kummerer, K. (2003). Significance of antibiotics in the environment. J Antimicrob Chemother 52:5–7. Volume 2 No.8, August 2012 ISSN 2224-3577
International Journal of Science and Technology
2012 IJST. All rights reserved Table 1: Antibiotic Susceptibility Pattern of Some Gram Negative Bacteria Isolated from Poultry Litter
Salmonella Key
SXT=Septrin, CH=Chloranphenicol SP= Sparfloxacin, CPX= Ciprofloxacin, AM = Amoxacillin, AU Augmentin, CN = Gentamycin, PEF =
Pefloxacin, OFX = Tarivid S=Streptomycin. R= resistant, I = intermediate, S= susceptibility, AB=Antibiotics.
Table 2 presents percentage of Antibiotic susceptibility pattern of some Gram negative bacteria isolated from poultry litter. The results revealed that Shigella responded totally (100%) to CH. with 33.3% response to CP, CPX, AM, and CN. The 66.7% response to SXT, AU, PEF, OFX and S. The table also shows that Salmonella had 33.3% response to SXT, CPX, PEF and OFX, 66.7% response to CP and CPX. Also observed was zero response to CH and AM. E. coli further showed 33.3% resistance to SXT and CPX, 66.7% to AM and zero to CH, CP, AU, CN, PEF, and S. Regression analysis also showed strong association (R=.516) between antibacterial resistivity and isolates. Table 2: Percentage of Antibiotic Susceptibility Pattern of Some Gram Negative Bacteria Isolated from Poultry
Salmonella R= .516 df=1
Key
SXT = Septrin, CH=Chloranphenicol, SP=Sparfloxacin, CPX= Ciprofloxacin, AM = Amoxacillin, AU Augmentin, CN = Gentamycin, PEF =
Pefloxacin, OFX = Tarivid and S=Streptomycin, AB= Antibiotics.
Table 3 shows bacteria isolated and their occurrence at different sites. The result revealed that GRA Gboko recorded highest with percentage
(28.8%), followed by Adekaa (25.6%) and University of Mkar community (24.8%), while Gboko central was least with (20.9%). Chi-square
results also showed a significant relationship (X2 = 5.871) between occurrence of bacteria and sample site.
Table 3: Bacteria Isolated and their Occurrence at Different Sites
Salmonella Total Occurrence
X2 =5.871 d

f (n-1) = 6 P>0.05
Key: UMC = University of Mkar Community

Source: http://ejournalofsciences.org/archive/vol2no8/vol2no8_8.pdf

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