ISSN: 2276-7762 Isoltion of High Antibiotic Resistnt Fecl Bcteri Indictors, Slmonell nd Vibrio Species from Rw Abttoirs Sewge in Peri- Urbn Loctions of Nirobi, Keny By Nymboy Rosemry Atieno Okemo Pul Owuor Ombori Omwoyo
Greener Journl of Biologicl Sciences ISSN: 2276-7762 Vol. 3 (5), pp. 172-178, July 2013. Reserch Article Isoltion of High Antibiotic Resistnt Fecl Bcteri Indictors, Slmonell nd Vibrio Species from Rw Abttoirs Sewge in Peri-Urbn Loctions of Nirobi, Keny 1 Nymboy Rosemry Atieno, * 2 Okemo Pul Owuor, 3 Ombori Omwoyo 1 Emil: mcjowi@gmil.com, Tel: +254721301212, 3 Emil: richrdombori@gmil.com, Tel: +254751839856 ABSTRACT Deprtment of Plnt nd Microbil Sciences, Kenytt University, P.O Box 43844-00100, Nirobi, Keny. *Corresponding Author s Emil: pulokemo@gmil.com, Tel: +254722942072 The reserch ws conducted to determine indictor orgnisms reltionship to specific humn pthogens nd ny presence of ntibiotic resistnce. Isoltionof indictor orgnisms,slmonell nd Vibrio species ws crried out using stndrd lbortory methods. Sensitivity to ntibiotics ws determined by the gr diffusion technique. The fecl bcteri lod ws found to be 6.2 x 10 6, 5.3 x 10 5, 2.5 x 10 4, 2.9 x 10 4, nd 5.0 x 10 6 CFU/100 ml for fecl streptococci nd 3.4 x 10 5, 4.1 x 10 3, 3.0 x 10 4, 2.7 x 10 3 nd 3.9 x 10 5 MPN/100 ml for fecl coliforms in cttle wstewter, cttle sludge, got wstewter, sheep wstewter nd mixture of got nd sheep sludge, respectively.fecl coliforms showed the highest resistnce with men resistnce frequency of 60.8% (±25.2), followed by Slmonell species t 51.5% (±26.6). Vibrio species showed the lowest men resistnce frequency t 41.6% (±24.8). There ws however no significnt difference (p=0.859) in resistnce mong Vibrio, Slmonell, FS nd FC isoltes t p>0.05.there is likelihood of slughterhouse nimls nd bcteri in the intestines of these nimls getting exposed to ntibiotics to which the bcteri develop resistnce which they cn pss to humn pthogens nd environmentl flor. Keywords: Antibiotic resistnce, Sewge, Pthogens nd Abttoirs. INTRODUCTION Enteric bcteri from humn nd niml feces cn be found in surfce wters; the fecl bcteri re brought into qutic environments minly through treted or untreted wstewter relese, surfce runoffs nd soil leching (Jmes et l., 2003). The presence of pthogenic enteric micro-orgnisms in qutic environments cn be source of disese when wter is used for drinking, recretionl ctivities or irrigtion. The snitry risk is incresed if the pthogenic enteric bcteri present in wters re ntibiotic resistnt becuse humn infections cused by such bcteri could be difficult to tret with drugs (Wenzel nd Edmond, 2009). In ddition, fecl bcteri might be ble to trnsmit ntimicrobil resistnce to utochthonous bcteri through lterl trnsfer, when the resistnce genes re crried by trnsferble nd mobile genetic elements such s plsmids nd thus contributing to the spred of ntimicrobil resistnce (Syhet l., 2005). In the livestock sector, different types of frm nimls re cpble of crrying wide rnge of zoonotic pthogens (Swi nd Schoonmn, 2012). Moreover, nimls brought for slughter into urbn res more often come from villges where pthogen control regimens re wek, un-coordinted nd often not vilble. Lck of veterinry services in these livestock rering res poses substntil risk of widespred occurrence of diseses in the livestock popultion nd concurrent humn exposure to these zoonotic disese gents (Swi nd Schoonmn, 2012). Livestock often ct s non-symptomtic crriers of humn pthogens such s E. coli 0157, Slmonell species nd Cmpylobcter which re rrely detected during routine nte-mortem exmintion nd their wstes my contin high concentrtions of the orgnisms. Animl wste cn therefore contminte humn nd niml drinking wter sources nd even soil when used s mnure (Christin et l., 2012). www.gjournls.org 172
Greener Journl of Biologicl Sciences ISSN: 2276-7762 Vol. 3 (5), pp. 172-178, July 2013. Fecl pollution of qutic resources my led to diseses in humns becuse of pthogens ssocited with this pollution or my ffect humn ctivities (Weryet l., 2010). Fecl indictor orgnisms re typiclly used to demonstrte the potentil presence or bsence of groups of pthogens ssocited with wstewter or sewge sludge (Ktor nd Rhodes, 2003). Fecl coliforms, totl coliforms, E. colind enterococci re the bcteril indictors currently used in the ssessment of wter qulity nd helth risks. These bcteril isoltes often occur in the feces nd intestines of wrm blooded mmmls including livestock. These indictor orgnisms re not pthogenic themselves. Orgnisms like fecl streptococci (FS), fecl coliforms (FC) nd E.coli reused s indictors of fecl contmintion of wters since they re esier nd reltively inexpensive to enumerte nd detect thn the pthogens themselves(cynthi et l., 2004). The incresed re-use of wstewter rises concerns bout the occurrence nd survivl of pthogens in the environment. For instnce, the survivl nd recovery of Slmonell species from surfce wter, wstewter nd bottled wter hve been investigted (Eton et l., 2005). Vibrio species hve lso been reported in drinking wter, surfce wter nd sewge. Slmonell species is dngerous wter borne bcteril pthogen in terms of humn helth nd diseses (Moniruzzmnet l., 2000). Vrious serotypes of Slmonell species hve reportedly been responsible for wter nd food borne epidemics in vrious countries (Rosminiet l., 2004)emphsizing the importnce of the pthogen s food sfety concern. Wter is lso n importnt source for humn infections with ntimicrobil resistnt Vibrio species (Byrugb, 2004) Severl previous studies hve concentrted on clinicl isoltes from humns, nimls nd some environmentl smples. Studies on wstewter nd sludge re limited. The contribution of bttoirs nd ssocited wstewters is rrely considered nd yet bttoirs re potentil sources of enteric bcteri tht could possess ntibiotic resistnce genes. In this study, indictor orgnisms nd pthogens such s Vibrio nd Slmonell species were isolted for their conventionl reltionships but most importntly ntibiotic resistnce nd environmentl impct. The primry objective of this study ws to determine the bundnce of fecl indictors nd detect Slmonell nd Vibrio species in the bttoir wstewters nd sludge. The study lso imed to determine the ntimicrobil resistnce of these bcteril isoltes. MATERIALS AND METHODS Smple collection nd preprtion A totl of thirty 100 ml smples of sludge nd rw niml wstewter were collected from the three slughterhouses in Nirobi County. Eighteen smples of wstewters (6 smples of got, sheep nd cttle wstewters ech) nd twelve smples of sludge (6 smples of cttle sludge nd mixture of got nd sheep sludge ech) were obtined from ll the three slughterhouses. Smples were collected three times between 9 nd 10 o clock in the morning in the month of Mrch 2012 nd April 2012 in sterile 200 ml glss bottles nd were trnsported to Kenytt University lbortory in n ice cooler box for nlysis. Wstewter smples tht were not nlyzed within four hours were stored t temperture of 4 C. All smples were nlyzed within 24 h. Isoltion nd identifiction of bcteril isoltes Stndrd microbiologicl methods (Mriit nd Okemo, 2009) were used to isolte nd enumerte FC (fecl coliforms) nd FS (fecl streptococci) nd to detect Vibrio nd Slmonell species in the smples of wstewters nd sludge. Pigmenttion of the colonies nd Grm s stining followed by stndrd biochemicl chrcteriztion [such s mortility, urese, H 2 S production, glucose fermenttion, indole, citrte utiliztion nd the cytochrome oxidse tests] were used to confirm the bcteril isoltes. The counts of FS were expressed in the CFU (colony forming units) nd tht of FC (fecl coliforms) were expressed in MPN (most probble number) nd quoted s mens±sd (stndrd devition). Antibiotic sensitivity test Sensitivity to ntibiotics ws determined by the gr diffusion technique recommended by the NCCLS (Ntionl Committee for Clinicl Lbortory Stndrds) (NCCLS, 2003) on Mueller-Hinton gr (Oxoid) using the following ntibiotic impregnted disks: mpicillin (25 µg); cotrimoxzole (25 µg); streptomycin (10 µg); chlormphenicol (30 µg); knmycin (30 µg); gentmicin (10 µg); penicillin G (1 unit); methicillin (5 µg); minocycline (30 µg); lincomycin (2 µg); erythromycin (15 µg); tetrcycline (25 µg) nd sulfmethoxzole (200 µg). www.gjournls.org 173
Greener Journl of Biologicl Sciences ISSN: 2276-7762 Vol. 3 (5), pp. 172-178, July 2013. Dt nlysis SPSS computer softwre version 16.0 ws used for dt entry nd sttisticl nlysis. Groups significnce tests were performed using student T-test nd one wy ANOVA (nlysis of vrince) t 5% significnce level nd P vlue of <0.05 ws considered s significnt. The mens were seprted using Tukey s test t 5% level. RESULTS Prevlence of fecl bcteri indictors Got nd sheep sludge hd the highest fecl coliform (FC) contmintion with men density of 3.9 x 10 5 (±3.5x10 5 ) MPN/100 ml followed by cttle wstewter with men density of 3.4 x 10 5 (±3.0x10 5 ) MPN/100 ml (Tble 1). The difference in men density of fecl coliforms from got nd sheep sludge nd men densities of fecl coliforms from cttle sludge, sheep nd got wstewters ws highly significnt (p=.004) t P<0.01. The overll men density of fecl streptococci (FS) ws higher (2.4 x 10 6 (±3.0x10 6 ) CFU/100 ml) compred to tht of the fecl coliforms (1.5 x 10 5 (±5.5x10 6 ) MPN/100 ml), however Student T-test showed no significnt difference. Cttle wstewter showed the highest fecl streptococci (FS) density with men density of 6.2 x 10 6 (±5.5x10 6 ) CFU/100 ml followed by got nd sheep sludge with men density of 5.0 x 10 6 (±4.5x10 6 ) CFU/100 ml. The difference in men density of fecl streptococci from cttle wstewters nd men densities of fecl streptococci from cttle sludge, sheep nd got wstewters ws highly significnt (p=.003) t P<0.01. Person correltion nlysis indicted tht prevlence of FS from ll sites showed significnt positive correltion with tht of FC (r = 0.931 t 0.01 level). Tble 1: Men fecl bcteri densities (men± stndrd devition) in 100 ml of the wstewters nd sludge from Kyole nd Kimko bttoirs in Nirobi, Keny Smple (n=30) Fecl streptococci Fecl coliforms Cttle wstewter 6.3 x 10 6b (±5.5x10 6 ) 3.4 x 10 5b (±5.5x10 6 ) Cttle sludge 5.3 x 10 5 (±5.5x10 6 ) 4.1 x 10 3 (±5.5x10 6 ) Got wstewter 2.5 x 10 4 (±5.5x10 6 ) 3.0 x 10 4 (±5.5x10 6 ) Sheep wstewter 2.9 x 10 4 (±5.5x10 6 ) 2.7 x 10 3 (±5.5x10 6 ) Got nd sheep sludge 5.0 x 10 6b (±5.5x10 6 ) 3.9 x 10 5b (±5.5x10 6 ) *Men 2.4 x 10 6 (±3.0x10 6 ) 1.5 x 10 5 (±5.5x10 6 ) Key: n-number of smples, fecl coliform density expressed in MPN/100 ml (most probble number) nd fecl streptococci density expressed in CFU/100 ml (colony forming unit). Sme letters indicte no significnt difference ccording to Turkey s Honest Significnce Difference (HSD) t 5% level. Detection of pthogens In Tble 2, the key tests for Vibrio species which include indole test, red slnt nd yellow butt in TSI (triose sugr iron) nd citrte utiliztion in ddition to Grm stined Grm negtive rods were observed. For the Slmonells, there ws mortility in SIM (sulphideindolemortility) indicting the presence of flgell in the isolted orgnism. The tests lso resulted in the clssicl presenttion of yellow butt, red slnt nd blckening in the tubes which is indictive of hydrogen sulphide production. It is lso worth noting tht these pthogenic bcteri were more frequently detected in smples tht showed high incidence of fecl bcteri indictors contmintion, for instnce cttle wstewter nd got nd sheep sludge. www.gjournls.org 174
Greener Journl of Biologicl Sciences ISSN: 2276-7762 Vol. 3 (5), pp. 172-178, July 2013. Tble 2: Biochemicl detection of Slmonell nd Vibrio species. Probble isolte SIM Ure TSI SCAN Peptone Cytochrome oxidse M I B 1 urese S B 2 B 3 C 1 C 2 G B 4 Vibrio species + + - - R Y - - + + + Slmonell species + - v - R Y + + v + + Key: v-vrible (+/-), M-mortility,I-indole, B 1 -blck, S-slope, B 2 -butt, B 3 -blck, C 1 -crck, C 2 -citrte utiliztion, G-gs, B 4 -blue, SCAN- simmons citrte gr, R-red, Y-yellow. Antibiotic resistnce ptterns All bcteril isoltes of fecl coliform origin (100%) were resistnt to lincomycin nd mpicillin, wheres only 30% were resistnt to cotrimoxzole nd minocycline (Tble 3). Most fecl coliform isoltes (50% nd bove) were however sensitive to chlormphenicol, cotrimoxzole, erythromycin, gentmicin, knmycin, streptomycin nd minocycline. Slmonell species on the other hnd showed high resistnce frequency to lincomycin (90%) nd methicillin (80%). Only 10% of the Slmonell strins were resistnt to chlormphenicol. The highest sensitivity frequency of the Slmonell isoltes ws observed with cotrimoxzole, chlormphenicol nd gentmicin (80%). The highest resistnce frequency mong FS species isoltes ws relted to mpicillin (90%), lincomycin nd methycillin (80%) nd tetrcycline (70%). Gentmicin nd chlormphenicol were found to be the most effective ntibiotics ginst FS with sensitivity frequency of 90%. With Vibrio species, the highest resistnce frequency ws observed with mpicillin nd sulfmethoxzole t 70% nd lincomycin t 90%. Agin, like with FS gentmicin nd chlormphenicl were found to be the most effective ginst Vibrio species. Tble 3: Sensitivity (%) of Fecl coliforms, Fecl streptococci, Slmonell spp. nd Vibrio spp. ginst 13 selected ntibiotics. FC Slmonell spp. FS Vibrio spp. Antibiotic (n=10) (n=10) (n=10) (n=10) S IR R S IR R S IR R S IR R Amp(25) 0.0 0.0 100.0 20.0 20.0 60.0 10.0 0.0 90.0 20.0 10.0 70.0 Chlo (30) 50.0 0.0 50.0 80.0 10.0 10.0 90.0 0.0 10.0 80.0 0.0 20.0 Cot (25) 70.0 0.0 30.0 80.0 0.0 20.0 70.0 0.0 30.0 50.0 10.0 40.0 Ery (15) 50.0 0.0 50.0 30.0 0.0 70.0 40.0 10.0 50.0 60.0 10.0 30.0 Gen (10) 50.0 0.0 50.0 80.0 0.0 20.0 90.0 0.0 10.0 70.0 0.0 30.0 Kn (30) 50.0 10.0 40.0 30.0 10.0 60.0 40.0 20.0 40.0 60.0 40.0 10.0 Linc (2) 0.0 0.0 100.0 10.0 0.0 90.0 10.0 10.0 80.0 0.0 10.0 90.0 Met (5) 20.0 0.0 80.0 20.0 0.0 80.0 20.0 20.0 80.0 20.0 30.0 50.0 Mino (30) 60.0 10.0 30.0 50.0 10.0 40.0 60.0 60.0 30.0 60.0 10.0 30.0 Pen (1) 30.0 0/00 70.0 50.0 20.0 30.0 30.0 30.0 70.0 60.0 10.0 30.0 Strep (10) 50.0 10.0 40.0 60.0 10.0 30.0 30.0 30.0 50.0 50.0 40.0 10.0 Sulf (200) 20.0 10.0 70.0 30.0 0.0 70.0 40.0 40.0 40.0 10.0 20.0 70.0 Tet (25) 20.0 0.0 80.0 10.0 20.0 70.0 30.0 30.0 70.0 30.0 10.0 60.0 Key: FC fecl coliform, FS Fecl Streptococci, S Sensitive, IR- Intermediry resistnt, R Resistnt, Amp- mpicillin, Linc-lincomycin, Pen-penicillin, Meth-methicillin, Ery-erythromycin, Tet tetrcycline, Cot-cotrimoxzole, Strep-streptomycin, Kn-knmycin, Gen-gentmicin, Sulf-sulfmethoxzole, Chlorm-chlormphenicol nd Mino-minocycline www.gjournls.org 175
Greener Journl of Biologicl Sciences ISSN: 2276-7762 Vol. 3 (5), pp. 172-178, July 2013. With regrd to cell wll morphology, Grm negtive bcteri (Fecl coliforms, Vibriospecies nd Slmonell species) ppered to be more resistnt to ll ntibiotics compred to the Grm positive bcteri (FS) in this study (Figure 1). Fecl coliforms ppered to hve the highest resistnce with men resistnce frequency of 60.8% (±25.2), followed by Slmonell species t 51.5% (±26.6). Vibrio species showed the lowest men resistnce frequency t 41.6% (±24.8). There ws however no significnt sttisticl difference (p=0.859) in resistnce mong Vibrio, Slmonell, FS nd FC isoltes. On the other hnd Vibrio species showed the highest sensitivity to ll the studied isoltes with men sensitivity frequency of 43.8% (±25.0), followed by FS t 42.3% (±26.2). Fecl coliforms were found to be the lest sensitive to ll the ntimicrobil gents used. There ws gin no significnt sttisticl difference (p=0.280) in sensitivity mong ll the bcteril isoltes. On the other hnd, there ws significnt difference (p=0.015) in intermediry resistnce to ntibiotics mong Vibrio ndfecl coliforms. Sensitivity of bcteril isoltes b b b Types of bcteril isoltes Sensitive Intermediry sensitive Resistnt Figure 1: Antibiotic sensitivity pttern of bcteril isoltes. Sme letters on similr brs indictes no significnt difference ccording to Turkey s HSD t 5% level. DISCUSSION A totl of 40 bcteril strins were isolted nd identified by their morphology nd biochemicl properties. Isoltes were identified up to genus level only. The five sources of bcteril contmintion chrcterized in this study were investigted becuse they differ in terms of the origin of the bcteri strin they relese in the environment (cttle, got nd sheep) nd in the expected exposure of these bcteri to ntimicrobil selective pressure. Fctors such s niml husbndry systems nd nture of psture grzing ptterns of individul nimls might hve contributed to the vrition in prevlence of microbes in different wstewter nd sludge smples. Fecl contmintion s indicted by the finding of FS nd FC could plce the environment t risk for hrboring microbes cpble of cusing humn diseses. This includes certin pthogenic strins of E. coli, Slmonell, Cmpylobcter, Aeromons nd protozo such s Girdi ll of which hve niml reservoirs (Snthiyet l., 2011). The trditionl multiple tube technique for FC detection remins useful especilly when the conditions do not llow the use of membrne filter technique. The simple nd time sving method of pour plting on stndrd Kenner Fecl (K F) streptococcus gr ws used to enumerte F S. Fecl streptococci were found mximlly in cttle wstewter nd miniml in got wstewter smples. Thus it ppers from these findings tht cttle could be the mjor crriers of these fecl bcteri. Presence of Slmonell sp. nd Vibrio sp.which re esily trnsmitted through wter re of gret concern. The sludge used s mnure is t risk of being contminted by Slmonell nd vibrio species nd thus poses gret risk to frmers nd consumers of vegetbles such s cbbges who use the produce to prepre
Greener Journl of Biologicl Sciences ISSN: 2276-7762 Vol. 3 (5), pp. 172-178, July 2013. slds. These bcteril isoltes re common intestinl bcteri of both nimls nd humns gut (Nbonitet l., 2011); however contmintion by Slmonell nd Vibrio species my lso come from public untreted wter pumped into slughterhouses or wter tken by nimls or cycling between the livestock nd their environment or even contmintion in feeds.gglirdi nd Krns, (2000) reported contmintion of wter used to irrigte vegetbles nd crops nd the mnure used s fertilizers with coliforms nd other enteric bcteri. In the present study, isoltes were most resistnt to lincomycin, mpicillin nd methicillin nd most sensitive to chlormphenicl, gentmicin nd cotrimoxzole. These results therefore show tht mpicillin, lincomycin nd methicillin re not effective to control these bcteri. Nipet l., (2011) observed 98% multiple resistnce to 2 7 different ntibiotics with 96.07% of the bcteril isoltes resisting mpicillin in the sme study. Bcteril resistnce to erythromycin, streptomycin, penicillin, mpicillin, moxicillin, knmycin, tetrcycline, oxytetrcycline nd chlormphenicol hve been reported (Roberts, 2011). The present study demonstrted tht bcteril isoltes were resistnt to ntibiotics commonly used s feed dditives (tetrcycline, streptomycin nd sulfonmides) or therpeutics (penicillin nd tetrcycline). Fecl coliforms showed the highest men resistnce frequency followed by Slmonell species. Vibrio species showed the lowest men resistnce frequency. Some Escherichi species nd Slmonell species possess cpsulr K nd Vi ntigens which protect them from ccess to ntimicrobils. High level ofprevlence of bcteril ntimicrobil resistnce similr to or higher thn those found in this study hve been reported in vrious qutic environments (Grci-Armiseet l., 2011; Mirnd et l., 2002; Zbigniew, 2005). The occurrence of ntimicrobil gents t low concentrtion vi leching or continued usge my led to the development of drug resistnce, which my led to resistnce trnsfer to pthogenic bcteri nd reduced efficcy of ntibiotic tretment for niml nd humn diseses (Tendenci nd De l Pen, 2002). Indeed the correltion between ntimicrobil use nd AR of commensl bcteri is well documented (Vn den Bogrd nd Stobberingh, 2000)nd we cn ssume tht the extent to which bcteril isoltes re exposed to ntibiotics before their relese in the environment could be the core reson for the levels of AR observed in this study. High resistnce to ntibiotics my lso be ttributed to severl other fctors; first it my be tht the source of drinking wter for the niml hd been polluted by ntibiotic gents; secondly introduction of ntibiotic supplemented commercil feeds my hve initited the resistnce s previously suggested (Le et l., 2005). High resistnce my lso be ttributed to the history nd dose of ntibiotic utiliztion, s well s the chemicl structure of the ntibiotics (Aminov nd Mckie, 2007). In Conclusion Vibrio nd Slmonell species were more frequently detected in smples which lso showed high incidence of fecl bcteri indictors. It will be however, highly specultive to conclude tht FS nd FC both correlte well with these pthogenic microorgnisms since the exct prevlence of the pthogenic microorgnisms ws not determined in the current study. There is likelihood of slughterhouse nimls getting exposed to ntibiotics to which they develop resistnce which they could spred to the environment. ACKNOWLEDGEMENT We thnk Kenytt University for giving us lbortory spce nd purchsing some of the regents used in this study nd prticulrly Mr. D. Ng ng, technicin in the Plnt nd Microbil Science lbortory, Kenytt University for technicl ssistnce. 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