World Journal of Medical Sciences 15 (1): 34-47, 2018ISSN 1817-3055© IDOSI Publications, 2018DOI: 10.5829/idosi.wjms.2018.34.47

Corresponding Author: T.E. Ayogu, Department of Microbiology Ebonyi State University Abakaliki, Nigeria.34

Antibiotic Susceptibility Pattern of Salmonella and Pseudomonas Species Isolated from Meat Market and

Ogoja Road Abattoir Effluents in Abakaliki Metropolis

T.E. Ayogu, J.O. Orji, E.C. Nwojiji, R.C. Umezurike and Ude Ude Ibiam

Department of Microbiology Ebonyi State University Abakaliki, Nigeria

Abstract: This work was aimed at determining the antibiotics susceptibility patterns of Salmonella andPseudomonas species isolated from abattoir effluents in Abakaliki Metropolis. A total of thirty (30) abattoireffluent samples (5 from each collection points viz; butchering point, rinsing point and discharging point) werecollected and transported to the microbiological laboratory unit of Ebonyi State University for bacteriologicalanalysis. Bacteria isolated were characterized using standard microbiological and biochemical techniques. Atotal of fifty (50) bacterial isolates (27 from meat market abattoir and 23 from Ogoja road abattoir) were isolatedfrom both meat market and Ogoja road abattoirs. Out of the 50 bacterial isolates, 30 (60 %) were Salmonellaspecies while 20 (40 %) were Pseudomonas species. A total of 15 Salmonella species and 12 Pseudomonasspecies were isolated from meat market abattoir, while a total of 15 Salmonella species and 8 Pseudomonasspecies were isolated from Ogoja road abattoir. Antibiotic susceptibility study on the organisms was carriedout using Kirby Bauer disc diffusion method according to Clinical Laboratory Standards Institute, (CLSI, 2005).The Salmonella species were most sensitive to gentamicin (90 %), followed by meropenem (80 %), ceftriaxone(50 %), ofloxacin (30 %), cefuroxime (20 %) and caftazidime (10 %) while amoxycillin (0.0 %), cefotaxime (0.0 %)and clindamycin (0.0 %) showed the least activity against the bacteria. Pseudomonas species were moresusceptible to meropenem (80 %), followed by gentamicin; (80 %), ofloxacin (60 %), ceftriaxone (20 %),ciprofloxacin (20 %), cefuroxime (20 %) and amoxycillin (20 %) while erythromycin (0 %), ceftazidime (0 %)cefotaxime (0 %) and chindamycin showed the least. The multiple antibiotics resistance (MAR) index ofSalmonella was 0.73 from the two abattoirs while that of the Pseudomonas from meat market abattoir was 0.79while that from Ogoja road was 0.82. This gave an indirect suggestion of the probable source(s) of theorganisms. The presence of these multidrug resistant bacterial strains isolated from abattoir effluents could bea vehicle for the spread of antibiotic resistance gene to other bacteria. Hence, there is need for adequatetreatment and safe disposal of abattoir effluent in Ebonyi State and also in Nigeria at large.

Key words: Antibiotic susceptibility Salmonella Pseudomonas species and Abakaliki

INTRODUCTION trash management; trashes are moreover discarded into

An abattoir is a particular facility accepted and [2]. Some pathogenic bacteria like Salmonella andregistered by the authoritarian power designed for Pseudomonas are most often isolated from waste waterassessment of animals, clean slaughtering, dispensation in unkept abattoir. as well as efficient conservation and storage of meat yield Salmonella is most extensively spread organism thatmeant for individual utilization. [1]. In addition, suitable can lead to food toxicity plus it is conveyed commonly viaservices to make certain secure discarding of abattoir infected cuisines and water [3].wastes in an approach that will not compose a probable Pseudomonas species are ubiquitous organismsrisk to community wellbeing, mammal wellbeing, as well as there within several different ecological setting, moreoverthe surroundings is measured extremely vital. The majority they can be secluded from diverse existing sources,of abattoirs within Nigeria contain no amenities meant for together with plant life, animals, as well as humans. Their

close by flow, thus leading to an environmental hazard

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capacity to live on smallest dietary necessities in addition Enterobacter aerogens, Salmonella, Escherichia colito stand a range of physical circumstances have permitted (including serotype 0157:H7), Shigella, Giardia lambliathe organisms to persevere mutually in society as well as parasite eggs, amoebic cysts, Aspergillus, Mucor,hospital settings. Saccharomyces andPenicillium species which are of

Pseudomonas is extremely everywhere within water public health importance [9]. These pathogens may makesystems and has inherent antimicrobial resistance owing threats to the community wellbeing by transferring insideto small surface covering permeability and an extrinsic earth water or outside water; wind or else vectors likeefflux force structure. animals, birds and arthropods can transmit diseases from

Pathogens can spread from animal to man by several these microorganisms [8]. Enteric bacteria from animaldifferent ways, for example via direct contact, feces can be gotten from surface waters; the fecal bacteriaconsumption of food or water that is contaminated, are brought into marine environments mostly throughindirect contact via objects that are contaminated and unprocessed wastewater discharge, plane runoffs andtransmission by vectors and by aerosols [4]. A study by earth leakage [10].[5] suggests that wild animals can transfer pathogens tohumans and other animals from abattoir waste by feeding Salmonella: This is a diverse organism. It is rod-shaped,on the same. Water contaminated with pathogens can Gram-negative, facultative anaerobe, non-spore formingalso cause infection in animals and humans drinking the and is mostly motile by way of peritrichous flagella,water or eating crops or foods contaminated by the water excluding S. Pullorum and S. Gallinarum, with no flagella.[6].Waste matter in the false intelligence is in commonmeasured to be water contamination, as well as the Classification of Salmonella: The Genus Salmonella isoutpouring from an abattoir. Waste matter sump push, forexample, pushes waste water from abattoir into a nearestwater body. In the perspective of waste watermanagement, waste matter that has been treated isoccasionally called secondary effluent, or treatedeffluent. The sludge sinks to the base, parting the peaksegment of water plain, open to be pumped back into theriver or be reused in the procedure once more [7].

An Abattoir is defined as a ground permitted plusregistered with the scheming power for clean slaughteringin addition to examination of animals, possessing as wellas efficient conservation with storage of meat yield forhuman use [8]. Within Nigeria, the abattoir manufacturingcenter is an essential part of the farm animal productionproviding household meat supply to above 150 millionpopulace and job opportunities for teaming populace [8].In Nigeria, adequate abattoir waste management is lackingin all public abattoirs such that huge solid wastes alongwith unprocessed effluents are general sites [2] unlike inurbanized countries where these amenities are sufficientlyprovided [3]. These abattoir wastes could be a source ofembarrassment since conventional methods of wastesupervision is ignored [6]. Adding to this, there could aswell be the company of pathogenic microorganisms, thesame as Enterobacter aerogenes, Hafniaalvei,Erwiniamallotivora, Edwardsiellaictaluri, Enterobacteramnigenus, Proteus miriabilis, Staphylococcus spp,rotaviruses, hepatatitis E virus, Pseudomonasaeruginosa, Enterobacter intermedius, Yersiniaaleksiciae, Serratiaodorifera, Enterobacter cloacae,

among the Domain Bacteria, Phylum Proteobacteria, ClassGamma Proteobacteria, Order Enterobacteriales, Family:Enterobacteriaceae and Genus Salmonella. There are twospecies under Salmonella namely, Salmonella entericaand Salmonella bongori [11]. The subspecies are furtherdivided into serotypes. Within Salmonella enterica, morethan 2000 serotypes have been identified; and theseaccount for about 99% of all the serotypes of Salmonella.

Current Nomenclature: Current Salmonella classificationscheme is based on biochemical character, DNAhomology and enzyme electrophoresis [12]. The taxonomyof the genus Salmonella developed from the first oneserotype-one species theory anticipated by Kauffmann onthe base of the serologic classification of somatic (O),flagellar (H) and capsular (Vi) antigens [13].

Identification of Salmonella: Since the importance ofSalmonella as a human pathogen a lot attempt has beencommitted to the growth and development of diversediscovery methods in diverse matrices along with medical,environmental, food and feed samples [14].

2 Molecular Detection Methods: Numerous molecularmethods are now projected for revealing Salmonellawithin foods as well as feeds. Choice of a particularprocess depends on attributes like reliability of themethod in detecting small amounts of Salmonella, cost,promptness, effortlessness of handling, likelihood ofease mechanization, lack of false-negative as well as

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false-positive results and international acceptability [15]. Transmission of Pathogens in the Abattoir Environment:Nevertheless, molecular methods used for recognition of Pathogens can spread from animal to man by severalSalmonella categorised as immunological and nucleic different ways, for example via direct contact,acid-based techniques [16]. These methods include consumption of food or water that is contaminated,enzyme-linked immunosorbent Assays (ELISA), enzyme- indirect contact via objects that are contaminated andlinked fluorescent assays (ELFA), immunomagnetic transmission by vectors and by aerosols [5]. A study byseparation (IMS), dip- stick and antibody microarray [17]. [6] suggested that wild animals can transfer pathogens toWhereas, Nucleic Acid-Based techniques used for humans and other animals from abattoir waste by feedingrecognition of Salmonella, are based on specific binding on the same. Water contaminated with pathogens canof flagellar antigens of the target organism to monoclonal also cause infection in animals and humans drinking theantibodies. PCR, DNA hybridization and DNA microarray water or eating crops or foods contaminated by the waterare examples of nucleic acid based methods used for [2]. An example of the latter was shown by [21], whererecognition of Salmonella [14]. they connected a multistate outbreak of disease caused

Antimicrobial Resistance of Salmonella: In addition to contaminated with the pathogen.its pathogenicity, there has been concern aboutantimicrobial resistance in Salmonella, which has led tofailure of treatment of Salmonellosis and other bacterialinfectious diseases [17]. Since the discovery of antibiotic-resistant bacteria in the 1940s, efforts are being made todevelop new antibiotics though at a very steady rate. Inthe present day, the unnecessary utilization of antibioticsand the lack of fresh effective chemotherapeutic agentson the sell have increased the trouble of antibioticresistance more than ever into a fast rising universalhealth disaster. The control of communicable diseases iscritically susceptible by the fixed increase in the numberof microorganisms that are resistant to antimicrobialagents. Infections by resistant pathogens badly influencedeath, cure costs, disease spread and length of sickness[18]. Drug resistance in bacterial pathogens likeSalmonella is mainly due to intensive utilization ofantimicrobial drugs in food-manufacturing animals andhumans [19].

Pseudomonas: Pseudomonas is a genus of Gram-negative,aerobic gamma proteo -bacteria, the family ofPseudomonadaceae that contains 191 validly describedspecies. The members of the genus show a big deal ofmetabolic variety and therefore are capable of colonizinga large range of niches. Their simplicity of culture in vitroand accessibility of a rising number of Pseudomonasstrain genome sequences has made the genus anoutstanding focal point for scientific research; the topstudied species consist of P. aeruginosa in its function asan opportunistic human pathogen, the plant pathogen P.syringae, the soil bacterium P. putida and the plantgrowth-promoting P. fluorescens. Since their prevalentrate in water and plant seeds such as dicots, thepseudomonads were observed early in the times past ofmicrobiology [20].

by EHEC O157:H7 to seeds of alfalfa sprouts

Transmission by Birds: Birds feeding from sewageoutfalls, rubbish tips or shellfish that’s been contaminatedcan pick up bacteria and then the bacteria can bedistributed to other places by the birds [22]. In a surveyof faecal samples from birds (mostly gulls) in 1997 theresults showed that a small percentage of the birdsincluded were carriers of Escherichia coli O157 [22]. In2006 [23], found, when investigating the source ofinfection for an outbreak of disease caused byEscherichia coli O157 in three humans, that isolates fromthe humans were identical to an isolate found in a samplefrom wild rooks’ faeces. Their results indicated thatindirect contact with faeces from wild birds can result ininfection with Escherichia coli O157 and that theinfection thereafter can carry on by person to persontransmission. It was suggested that the birds had pickedup the pathogen from faeces from livestock [23]. Severalstudies have shown that Salmonella spp. can be found inseveral different species of wild birds and that theytherefore can act as carriers of the bacteria [24]; [25]; [26];[27]. [24] found that the Salmonella spp. that was mostfrequently isolated from humans with diarrheal diseasewas found in wild crows in the same area. A study by [28]showed that contamination with Salmonella spp. andEscherichia coli in a water supply reservoir could beconnected to wild birds (gulls) roosting on the water.

The Marabou stork, Leptofiluscrumeniferus, is one ofthe largest and most common storks in Africa. It weighsapproximately five to six kilograms and has a wingspan upto four meters. Marabous are known scavengers and arereported to be omnipresent at abattoirs in some parts ofAfrica [29].

According to [30] Marabou storks often are in closecontact with humans for example at abattoirs. In the studyby [30] Marabou storks were euthanized and samples of

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faeces investigated for presence of Salmonella, however Escherichia coli resistant to several antibiotics haveno isolates were found to contain Salmonella. In a more previously been found in faeces from animals taken forrecent study by [27], the presence of pathogenic bacteria slaughter at abattoirs in Kampala [40]. Resistantin droppings from Marabou storks was investigated and Escherichia coli and Enterococcus spp. have previouslyit was found that 13 % of the droppings contained been isolated from wild birds and have been suggested asSalmonella, 14 % Escherichia coli and 9 % contained a danger to human health if spread to humans via faecalShigella. The study showed that Marabou storks can contaminated water [41].carry isolates of Salmonella, Escherichia coli andShigella. It was suggested that the bacteria originated Aim: This work is aimed at determining the antibioticsfrom the storks’ food and water sources [27]. The authors susceptibility patterns of Salmonella and Pseudomonasconcluded that faeces from the Marabou stork can be a species isolated from abattoir effluents in Abakalikipotential hazard to people’s health. Metropolis.

Earthworms: Earthworms have according to [31] been Specific Objectives:suspected to transmit animal and human pathogens. They To isolate and characterize Salmonella andare known sources of infection of parasites to poultry and Pseudomonas species isolates from abattoir effluentwild birds [32]. Microorganisms that are present in the in Abakaliki metropolis.environment of the worm are also often present in the To establish the prevalence of Salmonella andworm [31]. [33] found that Escherichia coli O157:H7 can Pseudomonas species in abattoir effluents.be found on and inside the earthworm To determine the susceptibility pattern ofDendrobaenaveneta after feeding in an environment Salmonella and Pseudomonas species to commonlycontaminated with Escherichia coli O157:H7. In a study used antibiotics. by [34], earthworms (Lampitomauritii) were placed in an To determine the Multi-Drug Resistance Index ofenvironment with material from several sewage treatment Salmonella and Pseudomonas species.facilities that were contaminated with Salmonella.Salmonella were found in the gut of the worms living in MATERIALS AND METHODSthe contaminated sewage waste up to 70 days after thestart of the study, though the study also showed that the Materials: The following materials used for this studylevels of Salmonella in the gut of the worms decreased were;over time. It also showed that Salmonella couldn’t befound in worms living in an environment with sewage Equipment/ Glassware: Sterile bijou bottle, sterilesludge mixed with cattle faeces and rice straw after 70 Universal container, Sterile Petri-Dishes, Sterile test tubes,days and the decrease of Salmonella levels in worms from Beaker, Measuring cylinder, Clean grease free slide, Wirethis group was also faster [34]. That the levels of loop, Marker, Masking tape, Cotton wool, Aluminum foil,Salmonella decreases when in presence of earthworms Conical flask (BEMA SCIENTIFIC & CHEMICALhave previously been showed in other studies, using NIG.Ltd).another earthworm named Eiseniafoetida [35]. The following instruments were used, Refrigerator,

Antibiotic Resistance in Abattoir Environments: burner, forceps, Incubator, Hot, air oven (GULFEXAntibiotic resistance measures that bacteria can refuse to MEDICAL &SCIENCTIFIC ENGLAND).accept the result of more than one antibiotic [36].Antibiotic resistance also leads to higher medical costs Reagent/Chemicals: The following reagents were usedand endangers the success of certain treatments [37]. It is 'distilled water, normal saline, sodium chloride, peptonewell known that animals can harbor antibiotic resistant water, iodine, acetone, ethanol, safranin, Kovacs Reagent.and zoonotic pathogens [4, 8, 38]. Multiple drugresistance has been suggested to be defined as when a Culture Media: The following culture media were used,bacterium has acquired resistance to one or more Nutrient agar, Nutrient broth, Mueller-Hinton agar,antibiotics in at least three antimicrobial categories [39]. Salmonella shigella agar, Triple Sugar and Iron (TSI)Pathogens that are resistant to antibiotics can be passed agar, Xylose lysine deoxycholate (XLD).(OXOID)on from animals to humans and vice versa [36]. As well

Microscope, Autoclave, Weighing balance, Bunsen

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Antibiotics: The following antibiotics were used State mostly populated and inhabited byCeftriaxone. 30µg Meropenem 10µg, Amoxycillin- indigenes and also people from other parts ofclavulanic acid 20µg, Ofloxacin 5µg, Erythromycin 15µg, Nigeria. Ebonyi State is surrounded in the east viaCiprofloxacin 5µg, Amoxycillin 20µg, Cefuroxime 30µg, cross River state, in the North via Benue State, in theCeftazidime 30µg, Ceforaime 30µg, Gentamicin 10µg and West via Enugu State as well as in the South via AbiaClindamycin 2µg. State. The climate is characterized by a hot dry period,

Method season is from May-October. The maximum temperatureResearch Region: This research was done in Abakaliki during the dry season is 37. 6°C while the minimumEbonyi State. Abakaliki town is a capital city of Ebonyi temperature is 27.1°C.

which stretches from November-April, while the rainy

Fig. 1: EbonyiState Showing the Position of Abakaliki.

Sterilization of Glassware: The glasswares were sterilized 5.3g was dissolved in 100ml of water and Mueller Hintonusing dry heat in a hot air oven at 160 degrees Celsius for agar, 3.8g was liquefied properly inside 500ml water and1 hour. then uncontaminated by autoclaving at 121°C for 15

Preparation of Culture Media: Every media used were were allowed to cool to 45°C before being dispensedprepared according to the producer’s instructions thus; aseptically into sterile Petri-dishes in 20ml volumes andFor nutrient agar, 2.8g was dissolved is 500ml of distilled then allowed to gel. For agar slants, the well dissolvedwater, for Salmonella- shigella agar, 63g was dissolved media were dispensed in 10ml volumes into Bijou bottlesin 1000ml of water, for Xylose lysine deoxycholate agar, and sterilized. They were then placed in a slanting

minutes in well corked conical flasks. The sterilized media

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position and allowed to cool and gel. For nutrient broth, Biochemical Tests: The following biochemical tests were1.3g was dissolved in 250ml of distilled water. This wasaseptically dispensed in 5ml volumes into test tubes,corked and sterilized in an autoclaves at 121°C for15minutes and allowed to cool.

Collection of Samples: A total of 30 abattoir effluentsamples were collected from two abattoirs, fifteen (15)each from Meat market abattoir and Ogoja road abattoirwithin Abakaliki metropolis. From the 15 samples(Abattoir effluents) collected from each abattoir, 5 werecollected from butchering point, rinsing point anddischarging point respectively. The effluents werecollected in sterile containers and were transported toEbonyi State University Microbiology Laboratorycomplex for bacteriological analysis, within 1hr ofcollection.

Cultivation of Abattoir Effluent: A loop full each of theabattoir effluents were first inoculated into sterile testtubes containing 5ml each of sterilized Nutrient broth alsothey were kept warm at 37°C up to 24 hours.

Isolation of Bacteria: A loop full each from the resultingbroths with turbidity be aseptically inoculated bystreaking on plates of Salmonella-Shigella agar for theseclusion of Salmonella specie and PseudomonasCetrimide selective agar plates used for the isolation ofPseudomonas. Inoculated Plates be kept warm at 37°C upto 18-24 hours.

Identification/ Characterization of the IsolatesGram Staining: Bacteria colonies were collected using asterile wire ring and emulsified in a plunge of sterilizedcondensed water placed on a glass glide to make a slenderspread. These were dried up by air and heat fixed bypassing the slide above a Bunsen burner flare to preventthe spread from being washed off during staining. Crystalviolet stain was used to fix the smear and it was allowedto stand for 60 seconds and it was later washed off withclean water. Lugol's iodine was then poured over thesmear and allowed to stand for 60 seconds and was rinsedoff with clean water. Acetone-alcohol was used todecolorize the smear and was washed off immediately.Then safranin was used to counter stain the smear andwashed off after about 30 seconds. The slide was placedon a draining rack to air dry before being examined underthe microscope using X40(to confirm the staining anddistribution of materials) and oil immersion XI 00.

carried out on the isolates.

Sulfide, Indole and Motility Medium (SIM): The center ofthe medium (which contains thiosulfate andtryptonedwater) was stabbed with a loop full of the testisolate. This was kept warm at 35°C up to 24-48hours andwas observed. Blackening of the medium indicated apositive test for H S (hydrogen sulphide) production and2

diffused growth from the line of inoculation spreadingoutward is indicative of a motile organism. Kovac'sreagent measurement of 0.5ml was added and the tubeshaken, a brick-red layer on the surface after 10 minutesindicated indole production [42].

ii. Nitrate Reduction Test: Nitrate broth in tubes wereinoculated with a heavy growth of the test isolates andincubated for 24-48hours at 37°C. Then, one drop each ofsulfanilic acid and alpha naphthalamine was added toeach broth. Red colouration indicated a positive test fornitrate reduction.

Sugar Fermentation Test: Using a sterile straightinoculation needle, a colony of the test isolate wascollected and the center of the TS1 agar (made of lactose,sucrose and glucose in ratio of 10:10:1) in a tube wasstabbed and also the surface of the agar was streaked.The tube medium remains red this indicated that there wasno fermentation. If the slant shows red and butt yellowcolour, this indicated that only glucose has beenfermented. If the slant and butt turn yellow, this willindicate that all 3 were fermented. And if there is blackcoloration, H S has been produced [42].2

Citrate test: A loop full of the isolate be inoculatedon Simmon’s citrate agar slope. Using wire loop that issterile, the slope was first streaked with the salinesuspension of the test organisms and the butt was thenstabbed. They were kept warm at 35°C up to 48 hours. Ifthe shown colour is blue, it indicates positive citrate testbut if no color change indicates that the citrate test isnegative [42].

Antibiotics Sensitivity Testing: Subsequent detection,the isolated microbes were subjected to antibioticsensitivity testing by disc diffusion technique usingcommercially available discs according to ClinicalLaboratory Standard Institute (CLSI).

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Preparation of McFarland StandardTurbidity: Standard DISCUSSIONTurbidity equivalent to 0.5 McFarland were organized byadding 1m of concentrated tetraoxosulphate (vi) acid to In this study, Salmonella spp. and Pseudomonas99ml of purified water and dissolve 0.5g of dehydrated spp. were isolated and identified in effluent samplesbarium chloride (BaCl 2H O) in 50ml of purified water in a collected from different points at both meat market and2 2

different reaction flax respectively. Barium chloride Ogoja road abattoirs (Table 1). The isolation ofsolution (0.6ml) was added to 99.4ml of the Salmonella spp. and Pseudomonas spp. from the twotetraoxosulphate (vi) acid solution in a different test tube abattoirs demonstrate the actuality that these Gramand the reaction mixture assorted well to form 0.5 negative bacteria are linked with abattoir effluents as theyMcFarland standard turbidity. Small portions of the turbid have been formerly isolated from other abattoir effluentssolution were transferred into a well capped test tubes in Nigeria by [43] and in Dourados, MatoGrosso do Sulrelated to the tube used initially for preparing the test State, Brazil by [44]. The detection of Salmonella spp. andmicroorganisms and were stored at room temperature [42]. Pseudomonas spp. from effluent samples collected fromAll the test bacteria were standardize individually before abattoirs is in agreement with the findings of [45] whichuse to 0.5 McFarlandstandards turbidity [6]. stated the presence of Pseudomonas aeruginosa,

Culture: A 0.lml suspension of each of the bacterial aureus from abattoir effluent samples collected fromisolates, equivalent to -0.5 McFarland standards was Ogbete in Enugu State. In a similar study, [46] reportedmade by diluting the culture with sterile NaCl and this was the presence of E. coli, Salmonella, Klebsiella,aseptically swabbed onto Mueller Hinton agar plates and Pseudomonas and Proteus in faeces from abattoir wastesallowed to stand for about 30 minutes to pre-diffuse. and products in Dutsin-Ma area of Katsina State, Nigeria.Different antibiotics types from the six classes of In another related study, [47] reported that Shigella andantibiotics (Macrolide, Quinolone, Tetracycline, Salmonella species were recovered from abattoir effluentAminoglycosidc, Cephalosporin, Cotrimoxazole,) were in Afikpo, South Eastern Nigeria. The presence of theseused. The antibiotic paper discs were aseptically placed opportunistic and pathogenic bacteria suggests theon the surface of the inoculated Mueller Hinton agar, presence of other pathogenic and opportunistic bacteria.using sterile forceps and incubated for 24hours at 37°C. From the result presented in Tables 2 – 5, it wasDiameters of the zones of inhibition were measured in shown that a total of fifty (50) bacterial isolates wereMillimeter and recorded, after growth/incubation. isolated from both meat market and Ogoja road abattoirs.

RESULTS abattoir while the least count (23) was obtained from

Result of Morphological, Microscopic and Biochemical microbial loads and can be attributed to the poor sanitaryCharacteristics of the Bacterial Isolates: Table 1 below and hygienic practices of the abattoirs’ management andrepresents the result of the cultural tests carried out on workers, the poor state of health of the slaughtered cowsthe isolates. It gives a summary of the morphological, and faecal contamination from the intestines of themicroscopic and biochemical characteristics of the slaughtered animals. As observed in the study location,bacteria isolates from meats market and Ogoja-road the entire environment of the abattoir are filled with fecalabattoirs. According to the different tests carried out materials from the animals that are being slaughtered andorganisms isolated were Salmonella spp.and also contaminants from the wastewater from thePseudomonas spp. butchering and dressing slabs in both abattoirs is rinsed

Escherichia coli, Salmonella spp and Staphylococcus

The highest count (27) was obtained from meat market

Ogoja road abattoir. These figures indicated very high

Table 1: Morphological, microscopic and biochemical characteristics of the bacteria isolates

Gram stain Biochemical tests Sugar tests

--------------------------- --------------------------------------------------------------------------------------------------- -------------------------------------------------------

Colony Cell Gram Methyl Voges Hydrogen Suspected

morphology Morphology reaction Catalase Motility Oxidase Citrate Indole red proskauer Urease sulphide Glucose Sucrose Lactose organisms

Translucent Colorless

Colony with

Black center Rod - + + - - - - - + + - - Salmonella spp.+

Bluish green pigment Rod - + + + + - - - - - - - Psuedomonasspp.-

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Table 2: Distribution of Salmonella Species Isolated from Various Parts of the Abattoir at Meat Market

Collection point No of samples collected No. of isolates Frequency %

Butchering point 5 5 33.3Rinsing point 5 5 33.3Discharging point 5 5 33.3

Total 15 15 100

This table shows that there was an equal distribution of Salmonella species in each point at meat market abattoir.

Table 3: Distribution of Pseudomonas Species Isolated from Various Parts of the Abattoir at Meat Market

Collection point No of samples collected No. of isolates Frequency %

Butchering point 5 3 25Rinsing point 5 4 33.3Discharging point 5 5 41.7

Total 15 12 100

This table shows an unequal distribution of Pseudomonas species in each point at meat market abattoir.

Table 4: Distribution of Salmonella Species Isolated from Various Parts of the Abattoir at Ogoja Road

Collection point No of samples collected No. of isolates Frequency %

Butchering point 5 5 33.3Rinsing point 5 5 33.3Discharging point 5 5 33.3

Total 15 15 100

This table shows that there was an equal distribution of Salmonella species in each point at Ogoja road abattoir.

Table 5: Distribution of Pseudomonas Species Isolated from Various Parts of the Abattoir at Ogoja Road

Collection point No of Samples Collected No. of Isolates Frequency %

Butchering point 5 2 25Rinsing point 5 3 37.5Discharging point 5 3 37.5

Total 15 8 100

This table shows an unequal distribution of Pseudomonas species in each point at Ogoja road abattoir.

into open drainage without any form of treatment. Related A total of 15 Salmonella species (Table 2) and 12study was reported by [48]. Pseudomonas species (Tables 3) were isolated from meat

Out of the 50 bacterial isolates, 30 (60 %) were market abattoir, while a total of 15 Salmonella speciesSalmonella species while 20 (40 %) were Pseudomonas (Table 4) and 8 Pseudomonas species (Table 5) werespecies. This high presence of Salmonella species in this isolated from Ogoja road abattoir. In the three collectionstudy is not surprising since Salmonella is reported to be points as shown in Table 2, there was an equalan environmentally relentless pathogen competent of distribution of Salmonella species which gave a highexisting and proliferating in varied surroundings [49]. frequency of Salmonella species 15(100 %) while Table 3Moreover, previous studies reported that Salmonella can shows an unequal distribution of Pseudomonas speciespersevere in the farm surrounding for extensive periods of at the three collection points where the highesttime owing to movement inside the farm from animals, occurrence was at the discharging point 15(41.7 %)human and livestock excrement, soil and plants [50]. followed by the rinsing point 4(33.3 %) and the least beingThe 60 % prevalence of Salmonella obtained in this study butchering point 3(25 %). This is in agreement with [45]is higher than the 33.3 % prevalence rate by [45]; 19.5 % who also had an equal distribution of Salmonella speciesby, [3] and 13.5 % by [8, 51] got 12.3 % from receiving at all the collection points and also stated the highwater bodies with 13.2 % from vegetables irrigated prevalence of Salmonella species and a low prevalence ofwith wastewaters from Gwagwalada abattoir, Pseudomonas species in their study. There was also anNigeria. In agreement with this study, is [48] which equal distribution of Salmonella species at the threereported 64 % prevalence of Salmonella from abattoir collection points at Ogoja road which shows a higheffluent in Afikpo. frequency occurrence (Table 4) while (Table 5) shows an

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Fig. 2: Percentage Susceptibility and Resistant Pattern of Salmonella spp Isolated from Meat Market

Fig. 3: Percentage Susceptibility and Resistant Pattern of Pseudomonas spp Isolated from Meat Market

Fig. 4: Percentage Susceptibility and Resistant Pattern of Salmonella spp Isolated from Ogoja road

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Fig. 5: Percentage Susceptibility and Resistant Pattern of Pseudomonas spp Isolated from Ogoja road

unequal distribution of Pseudomonas species where the being that Pseudomonas species isolated were fromdischarging point and rinsing point show the highest and different environments [53]. In consonance with thisequal distribution of 3(37.5 %) with the lowest occurrence study is [6] who recorded that Pseudomonas strainsat the butchering point 2(25 %). This result does not fully exhibited least resistance to imipenem, colistin sulphate,agree with the result of [51] who reported a 13.5 % meropenem and aztreonam. Most of all the bacterialSalmonella prevalence frequency in abattoir effluent isolates obtained in this work were highly susceptible tosamples. The reason for the slight difference may be as a gentamicin, meropenem and ofloxacin and this shows thatresult of the studied samples. The presence of Salmonella these antibiotics are still very effective in treatingand Pseudomonas spp. in the abattoir effluents is of infections caused by these microorganisms.public health concern. [52] reported that cattle are the Salmonella and Pseudomonas species isolatesreservoir of Salmonella that may be spread to humans obtained in this work were multidrug resistant as theirwith resulting illness. antibiotic resistance profiles showed that they were

In this study, Salmonella isolates were highly resistant to at least two classes of antibiotics. This is insusceptible to meropenem (80 %) and gentamicin (65 %), agreement with the report of [55] where all the bacterialas shown in Fig 2. Also Salmonella species isolates in isolates were multidrug resistant as they exhibitedFigure 4 were also susceptible to meropenem (80 %) and resistance to at least two classes of antibiotics. The resultgentamicin (90 %). In agreement with this study, is [48] presented in Table 6 shows the multiple antibioticswhich reported that Salmonella isolates were highly resistance (MAR) index of Salmonella and Pseudomonassensitive to Ciprofloxacin, Cetriaxone, Gentamicin and species isolated from Meat Market. It was revealed thatOfloxacin. [45] reported that Gentamicin, meropenem and the isolates had an average MARI of 0.78 (Salmonella)ofloxacin were the most active antibiotics against and 0.79 (Pseudomonas species). The result presented inSalmonella isolates. The Pseudomonas isolates were Table 7 shows the multiple antibiotics resistance (MAR)susceptible to meropenem (73.3 %) (Fig 3) and also in index of Salmonella and Pseudomonas species isolatedFig 5 they were susceptible to meropenem (80 %), from Ogoja Road. It was revealed that the isolates had anofloxacin (60 %) and gentamicin 80 %. This agrees with average MARI of 0.73 (Salmonella) and 0.82[45] who recorded that Gentamycin was the most effective (Pseudomonas species). The multiple antibiotic resistance(85.7 %) drug against Pseudomonas. Conversely, the (MAR) indices give an indirect proposal of the possibleantibiotic resistance patterns of Pseudomonas species source(s) of the organism. The MAR indices in this studyisolates obtained in this work contradicts the study done were greater than 0.20, this verified the statement of [6]in university of Pittsburgh hospital in Palermo, Italy, that the MAR index greater than 0.20 indicates that thewhere Pseudomonas isolates obtained from the patients organisms must have begun from an environment wherein intensive care unit were resistant to Ofloxacin reason antibiotics are frequently used as also testified by [6, 56]

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Table 6: Multiple Antibiotics Resistance (MAR) Index of Salmonella and Pseudomonas species Isolated from Meat Market

SAL. n = 20, Ps. n = 15-------------------------------------------------------------

Isolate Code MARI Isolate Code MARI

SAL 1 0.9 Ps 1 0.9SAL 2 0.6 Ps 2 0.4SAL 3 0.8 Ps 3 0.8SAL 4 0.8 Ps 4 0.9SAL 5 0.9 Ps 5 0.8SAL 6 0.9 Ps 6 0.7SAL 7 0.6 Ps 7 0.8SAL 8 0.9 Ps 8 0.9SAL 9 0.8 Ps 9 0.9SAL 10 0.7 Ps 10 0.9SAL 11 0.9 Ps 11 0.9SAL 12 0.8 Ps 12 0.6SAL 13 0.9 Ps 13 0.8SAL 14 0.8 Ps 14 0.8SAL 15 0.8 Ps 15 0.8SAL 16 0.5SAL 17 0.6SAL 18 0.7SAL 19 0.8SAL 20 0.9

Table 7: Table 6: Multiple Antibiotics Resistance (MAR) Index ofSalmonella and Pseudomonas species Isolated from Ogoja Road

Sal. n = 10, Ps n = 5-------------------------------------------------------------

Isolate Code MARI Isolate Code MARI

SAL 1 0.7 Ps 1 0.8SAL 2 0.5 Ps 2 0.9SAL 3 0.9 Ps 3 0.8SAL 4 0.8 Ps 4 0.8SAL 5 0.8 Ps 5 0.8SAL 6 0.6SAL 7 0.8SAL 8 0.8SAL 9 0.6SAL 10 0.8

KEY: SAL = Salmonella, Ps = Pseudomonas

and [57]. Thus, from the result of the multiple antibioticresistance indexes in this work it can be stated that thesepathogens might have originated where these antibioticsare used.

CONCLUSION

In conclusion, the research has described thebacterial profile and multidrug resistance traits ofSalmonella and Pseudomonas species from abattoireffluents in meat market and Ogoja road abattoirs all

located in Abakaliki Ebonyi State as a serious publichealth concern. This study observed that the sanitaryconditions of the environments where these two abattoirsare located are very poor. The presence of these multidrugresistant bacteria in abattoir effluents will pose a seriouspublic health problem if not properly treated before beingdischarged into the environment. The public healthconsequences could be grave especially when they enterwater bodies which are commonly used for domesticpurposes. Thus, it is very imperative that abattoireffluents be properly treated before being discharged intothe environment. Most of all the bacterial isolatesobtained in this work were highly susceptible togentamicin, meropenem and ofloxacin and this shows thatthese antibiotics are still very effective in treatinginfections caused by these microorganisms.

Recommendations:Abattoir effluents which are channeled into waterbodies should be decontaminated before discharginginto the waters and the animals examined beforeslaughtering.In order to avoid serious public health problem, thepublic should be educated on;

Proper hygiene.Increase in hand washing techniques.Proper treatment of water obtained from variouswater bodies before domestic use.Proper washing of vegetables and fruits beforeconsumption.

Therefore, the importance of adopting suitableabattoir wastewater management procedures to preventthe chances of contaminating water bodies and groundwater is recommended.

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