research article isolation of environmental bacteria from surface...

Research ArticleIsolation of Environmental Bacteria from Surface andDrinking Water in Mafikeng South Africa and CharacterizationUsing Their Antibiotic Resistance Profiles

Suma George Mulamattathil1 Carlos Bezuidenhout2

Moses Mbewe1 and Collins Njie Ateba3

1 Department of Water and Sanitation University of Limpopo Turfloop Campus Private Bag X 1106 Sovenga 0727 South Africa2 School of Biological Science North-West University Potchefstroom Campus Private Bag X 6001 Potchefstroom 2520 South Africa3 Department of Biological Sciences School of Environmental and Health Sciences North-West University Mafikeng CampusPrivate Bag X2046 Mmabatho 2735 South Africa

Correspondence should be addressed to Suma George Mulamattathil sgmulamattathilgmailcom

Received 15 January 2014 Revised 22 May 2014 Accepted 16 June 2014 Published 6 July 2014

Academic Editor Mariela A Segura

Copyright copy 2014 Suma George Mulamattathil et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The aim of this study was to isolate and identify environmental bacteria from various raw water sources as well as the drinkingwater distributions system in Mafikeng South Africa and to determine their antibiotic resistance profiles Water samples fromfive different sites (raw and drinking water) were analysed for the presence of faecal indicator bacteria as well as Aeromonas andPseudomonas species Faecal and total coliforms were detected in summer in the treated water samples from the Modimola damand in the mixed water samples with Pseudomonas spp being the most prevalent organismThemost prevalent multiple antibioticresistance phenotype observed was KF-AP-C-E-OT-K-TM-A All organisms tested were resistant to erythromycin trimethoprimand amoxicillin All isolates were susceptible to ciprofloxacin and faecal coliforms and Pseudomonas spp to neomycin andstreptomycin Cluster analysis based on inhibition zone diameter data suggests that the isolates had similar chemical exposurehistories Isolates were identified using gyrB toxA ecfX aerA and hylH gene fragments and gyrB ecfX and hylH fragments wereamplifiedThese results demonstrate that (i) the drinking water fromMafikeng contains various bacterial species and at times faecaland total coliforms (ii) The various bacteria are resistant to various classes of antibiotics

1 Introduction

Water is considered a vehicle for the propagation and dis-semination of human associated bacteria [1] Safe drinkingwater is a fundamental human right and if contaminatedwith opportunistic pathogenic environmental bacteria itmay have health implications for consumers [2 3] Humanhealth should therefore be protected by preventing microbialcontamination of water that is intended for consumption [4]In rural communities untreated surface water from riversdams and streams is directly used for drinking and otherdomestic purposes [5] These unprotected water sourcescan be contaminated with microbes through rainfall run-off and agricultural inputs mixing with sewage effluents and

faeces from wild life [6 7] which render them unacceptablefor human consumption Faecal coliforms Aeromonas andPseudomonas are used as indicators of faecal contaminationin water [8] and the presence of these pathogens may havesevere health implications on consumers especially those thatare immunocompromised [5 9 10]

South Africa is a semiarid country with very low rainfalland high evaporation [11] and it has a scarcity of fresh watersystems due to the highly variable and spatial distributionof rainfall [12] Moreover safe drinking water is frequentlyused for unsustainable nondrinking applications such as irri-gation toilet and urinary flushing [12] and general cleaningTo manage the existing water resources and to address someof the challenges associated with water shortages in South

Hindawi Publishing CorporationJournal of PathogensVolume 2014 Article ID 371208 11 pageshttpdxdoiorg1011552014371208

2 Journal of Pathogens

Africa and the world at large waste water reuse can forman important component of water demandmanagement [13]but this waste water reuse may affect the quality of drinkingwater if proper treatment procedures are not implementedWhereas waste water reuse has been extensively implementedin some European and African countries [13] yet in SouthAfrica only a few waste water reuse schemes have beendocumented [14 15] and there is limited implementation ofthis alternative in communities

Alarming increases in the consumption of antibioticsthrough human therapy and agricultural processes have beenreported [16 17] and this extensive usage in both humanand animal medicine has resulted in the development ofantibiotic-resistant bacteria which affect the treatment ofinfections [18 19] Antibiotic resistance has therefore becomea major public health issue [20] and its presence in wastewater surface water and drinking water is well documented[19ndash22] The hazard associated with the pathogenicity ofmicrobes is aggravated by its ability to resist destructionby antibiotics Biological treatment processes in the wastewater treatment plants may result in a selective increaseof antibiotic-resistant bacteria and therefore increase theoccurrence of multidrug-resistant organisms [23] Althoughmicroorganisms in drinking water are reduced by chlorina-tion they may survive the treatment process and enter thedistribution system [1] Moreover the presence of antibioticresistance in microorganisms has been previously reported[24ndash26] Considering the fact that the public health of acommunity may be related to the quality of treated wastewater supplied and that public health can be protected byreducing the pathogenic microorganisms in drinking waterthe present study was designed to isolate environmentalbacteria from surface and drinking water in Mafikeng andidentify the Pseudomonas and Aeromonas species usingpolymerase chain reaction (PCR) A further objective wasto characterize the isolates using their antibiotic resistanceprofiles

2 Materials and Methods

21 Study Area Water samples were collected from fivesampling points around Mafikeng namely both raw andtreated (drinking) water from a karstic groundwater sourcethe Molopo Eye both raw water and treated (drinking)water from the Modimola dam and finally mixed watertreated water from both sources mixed in the Signal hillreservoir and distributed to some areas in the city Thesesampling pointswere chosen for the study becausewater fromMolopo eye andModimola dam after purification is used forhuman consumption and for recreational agricultural andindustrial purposes As few small scale of farmers live nearthese two water resources and the Modimola Dam receivestreated sewage effluent from theMmabatho sewage treatmentplant which is the major source of pathogens it is thereforeimportant to investigate the microbiological quality of waterat these points

22 Sampling Raw and treated water samples were collectedduring a one-year period in February April July and

October to cover the four different seasons Water samplesfrom the Molopo eye and Modimola dam were collectedaseptically in sterile 500mL Duran Schott glass bottles fromdifferent sampling points by directly dipping the bottles intothe surface of the water Purifiedwater samples were collecteddirectly in to the sterile bottles from the tap after letting thetap run for a minute The samples were labelled properly andtransported on ice to the laboratory for analysis Aliquotsof the samples were used for selective isolation of faecalcoliforms total coliforms Aeromonas Pseudomonas andheterotrophic bacteria based on standard microbiologicalprocedures [27]

23 Isolation Purification and Characterization ofPlanktonic Bacteria

231 Isolation by Membrane Filtration For all the samplesthree volumes of 100mL were filtered through 045120583m pore-sized filter (cellulose nitrate membranes Whatman Labo-ratory Division Maidstone England) using a water pump(model Sartorius 16824) These membranes were asepticallyplaced up on plates with appropriate selectivemedia ensuringthat no air bubbles were trapped The selective media usedare as follows mFC agar used as a selective medium forfaecal coliforms mEndo for total coliforms nutrient agar forheterotrophic bacteria and Aeromonas selective medium forAeromonas and Pseudomonas (Biolab Merck South Africa)All the media were prepared according to the manufacturersrsquoinstructions (Biolab Merck South Africa) Each samplewas analysed in triplicate In order to isolate heterotrophicbacteria 1mL of the treated water samples was spread ontothe nutrient agar plates Water samples from Modimola damand Molopo eye were serially diluted and 1mL of the 5 foldserial dilutions was spread on to the nutrient agar plates

The plates were incubated at 37∘C except for mFC agarwhich were incubated at 45∘C for 24 hours The colonieswere enumerated characterized and recorded The resultswere expressed as the number of faecal coliforms total col-iforms Pseudomonas and Aeromonas in 100mL of water andheterotrophic bacteria in 1mL of water Blue colonies frommFC agar (presumptive coliforms) metallic-sheen coloniesfrom mEndo agar (presumptive total coliforms) and yellow(presumptive Aeromonas) and green colonies (presumptivePseudomonas) from Aeromonas selective media were pickedfor further work (Biolab Catalogue)

232 Purification of Colonies Colonies were purified bytwice subculturing using the streaking plate method Youngcultures were used for Gram staining and all isolates wereidentified as Gram-negative Bacilli All the Gram-negativeisolates were subjected to primary and secondary biochemi-cal identification testsThe bacteria that were picked to createantibiogramswere streaked on to nutrient agar slants tomakesample cultures and for PCR purpose

24 Antimicrobial Susceptibility Testing An antibiotic sus-ceptibility test was performed using the Kirby-Bauer diskdiffusion method [28] The following antibiotic discs (Mast

Journal of Pathogens 3

DiagnosticsUK) at the final concentrations that are indicatedwere used ampicillin (AP) minus10 120583g cephalothin (KF) 5 120583gstreptomycin (S) 10 120583g erythromycin (E) 15120583g chloram-phenicol (C) 30 120583g neomycin (NE) 30 120583g amoxycillin (A)10 120583g ciprofloxacin (CIP) 5 120583g trimethoprim (TM) 25 120583gkanamycin (K) 30 120583g and oxytetracycline (OT) 30 120583g Theseantibiotics were chosen because they are either used in bothhuman medicine and animal veterinary practice or becauseprevious studies have reported microbial resistance to them[29]

Three colonies were picked from each sample and eachcolony was transferred in to 3mL of sterile distilled waterto prepare bacterial suspension Aliquots of 100 120583L fromeach suspension were spread-plated on Mueller-Hinton agarplates Antibiotic discs were applied on to the plates usingsterile needles and the plates were incubated at 37∘C for 24hours [30] After incubation the antibiotic inhibition zonediameters (IZD) were measured Results obtained were usedto classify isolates as being resistant intermediate resistantor susceptible to a particular antibiotic using standard ref-erence values according to National Committee for ClinicalLaboratory Standards [30] now Clinical and LaboratoryStandards Institute (CLSI) Multiple antibiotic resistance(MAR) phenotypes were generated for isolates that showedresistance to 3 or more antibiotics

25 Primary Identification Tests

251 Triple Sugar Iron (TSI) Test Triple sugar iron (TSI) agaris a differential medium obtained from Biolab Merck (SouthAfrica) with the substrates glucose sucrose and lactose atsample concentrations of 01 10 and 10 respectively Itcan distinguish between a number of Gram-negative entericbacteria based on their physiological ability (or lack thereof)to metabolize lactose andor sucrose conduct fermentationto produce acid produce gas during fermentation andgenerate H

2S The media were prepared according to the

manufacturerrsquos instructions Aliquots were placed in testtubes and autoclaved Once out of the autoclave the tubeswere placed on a rack and clamped so that the tubes (withliquid medium in them) have a 3 cm slant with a 2 to 3 cmbuttTheywere allowed to cool down until they become solidThe slants are inoculatedwith a pure culture by streaking overthe entire surface of the slant (zigzagging to cover the surface)and then stabbing deep into the butt Incubation was at 37∘Cfor 24 hours If only glucose is fermented acid is producedin the butt and it will turn yellow However if either sucroseor lactose is fermented sufficient fermentation products willbe formed to turn both the butt and the slant yellow If gas isformed during the fermentation it will be shown in the butteither as bubbles or as cracking of the agar If no fermentationoccurs the slant and butt will remain red The medium alsocontains ferrous sulphate If the bacterium forms H

2S this

chemical will react with the iron to form ferrous sulphidewhich is seen as a black precipitate in the butt (a black butt)

252 Oxidase Test This test was performed using the testoxidase reagent (PL390) from Mast Diagnostics (NestoWirral UK) in accordance with themanufacturerrsquos published

protocol A well-isolated pure colony was placed on a filterpaper using a sterile wire loop A drop of test oxidasereagent was added on to it and mixed After 30 secondsthe filter was observed for a colour change with oxidasepositive isolates producing a purple colour being taken aspresumptive Aeromonas and Pseudomonas isolates Oxidasenegative colonies were colourless and were presumptivelyconsidered to be E coli

26 Secondary Identification Tests

261 Analytical Profile Index (API) 20E Test The API 20Etest was performed in accordance with the manufacturerrsquosprotocol (BioMerieux 69280 Marcy IrsquoEtoile France) and theorganisms were identified to species level using API software

262 Haemolysis on Blood Agar Haemolysis on blood agar(BiolabMerck SA) supplementedwith 5 (vv) sheep bloodwas determined after the incubation of the plates at 37∘C for24 hours Haemolysis is determined by streaking for isola-tion on a blood agar plate After incubation overnight themedium is inspected for signs of alpha- or beta-haemolysisIf the medium is discoloured or darkened after growththe organism has demonstrated alpha-haemolysis If themedium develops clear halo under growth the organism isbeta-haemolytic No discernible change in the colour of themedium constitutes gamma haemolysis

27 Extraction of Genomic DNA and PCR for the Identificationof Culture Species DNA from the isolates was extracted usingthe peqGOLD (PEQLAB Biotechnologie GmbH 12-3450)bacterial DNA extraction kit according to the manufacturerrsquosprotocol The concentration of the extracted DNA in solu-tion was determined spectrophotometrically (NanoDropND1000 Thermo Scientific USA) The integrity of the purifiedtemplate DNAwas assessed by conventional 1 (wv) agarosegel

271 Identification of the Isolates by PCR Assays The identi-ties of the presumptivePseudomonaswere confirmed throughamplicons of gyrB 222 [31] toxA 367 [32] and ecfX 528[33] gene fragments respectively The identity of Aeromonasspecies was determined by screening them for the presence ofspecific virulent genes aerolysin (aerA) [34] and haemolysin(hylH) [35]

PCRswere performed using oligonucleotide primer com-binations and cycling conditions that appear in Tables 1 and2 Amplifications were performed using a Peltier ThermalCycler (model-PTC-220DYAD DNA ENGINE MJ ResearchInc USA)The reactionswere prepared in 25 120583L volumes thatconstituted 1 120583g120583L of the template DNA 50 pmol of eacholigonucleotide primer set 1x PCR master mix and RNasefree water All PCR reagents used were Fermentas USAproducts supplied by Inqaba Biotechnological Industries PtyLtd Pretoria South Africa All PCR products were stored at4∘C until used for further analysis

272 Electrophoresis of PCR Products PCR products wereseparated by electrophoresis on 2 (wv) agarose gel


Table 1 Oligonucleotide primers that were used for specific detection of Pseudomonas species

Primer Oligonucleotide sequence (51015840-31015840) Target and size (pb) PCR cycling conditions

ECF1ECF2

ATGGATGAGCGCTTCCGTGTCATCCTTGCCTCCCTG ecfX (528)

35X 94∘C for 45 s58∘C for45 s72∘C for 1m

GyrPA-398GyrPA-620

CCTGACCATCCGTCGCCACAACCGCAGCAGGATGCCGACGCC gyrB (222)


ETA1ETA2

GACAACGCCCTCAGCATCACCAGCCGCTGGCCCATTCGCTCCAGCGCT toxA (367)


Initial denaturing step of 95∘C for 5min and final strand extension of 72∘C for 5min

Table 2 Oligonucleotide primers that were used to detect virulence genes in Aeromonas species

Genes Oligonucleotide sequence (51015840-31015840) Target gene and size (bp) PCR cycling conditions

aer A Aer 2F AGCGGCAGAGCCCGTCTATCCAAer 2R AGTTGGTGGCGGTGTCGTAGCG aerA (416)

30X 95∘C for 2m55∘C for 1m72∘C for 1m

hyl H Hyl 2F GGCCCGTGGCCCGAAGATGCAGGHyl 2R CAGTCCCACCCACTTC hylH (597)



Electrophoresis was conducted in a horizontal PharmaciaBiotech equipment system (model Hoefer HE 99X Amer-sham Pharmacia Biotech Sweden) for 2 h at 60V using 1xTAE buffer (40mM Tris 1mM EDTA and 20mM glacialacetic acid pH 80) Each gel contained a 100 bp DNAmolecular weight marker (Fermentas USA) The gel wasstained in ethidium bromide (01 120583gmL) for 15min andamplicons were visualized under UV light A Gene GeniusBioimaging system (Syngene Synoptics UK) was used tocapture the image using GeneSnap (version 30701) software(Syngene Synoptics UK)

28 Statistical Analysis Cluster analysis based on the antibi-otic inhibition zone diameter data of different organismsisolated from different sites was determined using Wardsalgorithm and Euclidean distance of Statistica version 7

3 Results

31 Occurrence of Coliform Bacteria and Aeromonas andPseudomonas Species inWater Theprimary aim of this studywas to determine the levels of environmental bacteria fromsource and drinking water from Mafikeng Raw water fromModimola dam and Molopo eye treated water from thesetwo sites and mixed water were analysed for the presence oftotal coliforms faecal coliforms heterotrophic bacteria andAeromonas and Pseudomonas species Table 3 shows the aver-age number of different organisms isolated from the varioussites during summer and winter Heterotrophic bacteria wereisolated from all the sampling sites in both seasons and theiroccurrence was high Moreover Pseudomonas species faecaland total coliforms were the most prevalent during all theseasons in both raw and treated water from Modimola dam

However Aeromonas species were isolated only from the rawwater samples not from treated water from all the sites Thenumbers of the different organisms isolated were higher insummer than in winter

No total coliforms were observed in treated water fromthe Molopo eye Pseudomonas species were isolated in largenumbers fromModimola dam both in raw and treated waterfrom this site

32 Biochemical Tests Used to Identify the Isolates API testsresults are listed in Table 4 Various species were isolatedfrom the water sources particularly from the untreated waterThe diversity of bacteria from this source was greater thanfrom the treated water Pseudomonas spp were isolated fromthe Modimola dam (untreated as well as treated) waterthe Molopo eye (untreated only) and the mixed waterAeromonas sppwere isolated from the untreated aswell as themixed water Aeromonas and Pseudomonas species isolatedin this study were 120573-haemolytic and indicated potentialpathogenicity

33 Antibiotic-Resistant Data of Different Isolates from Drink-ing Water Isolates were subjected to an antibiotic suscep-tibility test using 11 different antibiotics from which theirantibiotic resistance profiles and multiple antibiotic resis-tance phenotypes were compiled The results obtained aredepicted in Tables 5 6 7 and 8 The results revealed that alarge proportion of the environmental isolates were resistantto erythromycin followed by trimethoprim and amoxicillinNone of the isolates were resistant to ciprofloxacin andonly very few isolates from Modimola dam were resistantto streptomycin and neomycin Despite the fact that allisolates from Molopo eye treated water from Molopo eye


Table 3 Average number of microorganisms isolated

Site Seasons FC TC H Ae Ps

Molopo eye (ME) Summer 30 40 gt100 42 gt100Winter 50 40 5 28 gt100

Modimola dam (MD) Summer gt100 gt100 gt100 gt100 gt100Winter 35 60 20 6 gt100

Treated water ME Summer 15 0 17 0 0Winter 0 0 7 0 0

Treated water MD Summer 15 6 20 0 gt100Winter 0 0 7 0 gt100

Mixed water Summer 5 5 10 0 0Winter 0 0 3 0 1

FC faecal coliforms TC total coliforms H heterotrophic bacteria and Ae Aeromonas

Table 4 Identification of the isolates from the treated (drinking)and source water using biochemical tests

Site API 20E (presumptive isolates)

Modimola dam (MD)untreated water

Pseudomonas aeruginosaPseudomonas luteolaAeromonas hydrophilaE coliSerratia odoriferaSerratia liquefaciensProteus vulgarisProvidencia rettgeri1Chryseobacterium meningosepticum

MD treated water

Pseudomonas aeruginosaPseudomonas luteolaSerratia odoriferaE coli

Molopo eye (ME) untreatedwater

Serratia odoriferaSerratia liquefaciensAeromonas salmonicida sppPseudomonas oleovoransChryseobacterium meningosepticumSalmonella choleraesuisEnterobacter asburiae

ME treated water Serratia liquefaciensMyroides spp

Mixed water

Serratia liquefaciensSerratia odoriferaAeromonas salmonicida sppPseudomonas aeruginosaMyroides spp

and Modimola dam and mixed water were susceptible toneomycin only a small proportion (20) from Modimoladam was resistant to this antibiotic Multiple antibiotic-resistant (MAR - 3 to 8 antibiotics) bacteria were commonamong the isolates

Faecal coliforms from all sites were susceptible to strepto-mycin neomycin and ciprofloxacin respectivelyThe highestnumber of bacteria was resistant to erythromycin How-ever two isolates from Modimola dam were susceptible toerythromycin Heterotrophic bacteria were isolated from all

sites and were also resistant to several antibiotics Isolatesfrom all sites were susceptible to streptomycin neomycinand ciprofloxacin Aeromonas species isolated were all resis-tant to erythromycin All of these species were susceptibleto ciprofloxacin However isolates from Molopo eye weresusceptible to kanamycin neomycin and streptomycin whileisolates from the Modimola dam were resistant to mostantibiotics All these isolates were resistant to ampicillinoxytetracycline trimethoprim and amoxicillin There weresingle isolates from Pseudomonas spp for Modimola damMolopo eye (untreated water sources) and the Modimoladam treated water and mixed treated water All the isolateswere resistant to up to eight antibiotics but all were suscepti-ble to streptomycin neomycin and ciprofloxacin

331 Predominant Multiple Antibiotic-Resistant (MAR) Phe-notypes Observed among the Bacterial Groups Differenttypes of multiple antibiotic resistance patterns were observedamongst all the bacterial groups isolated from the varioussites In some cases some of the isolates were resistance toup to ten different antibiotics The predominant antibiotic-resistant phenotypes that were obtained for the groups fromdifferent sites are depicted in Table 9 Similar types of MARphenotypes were observed in isolates from groups

34 Cluster Analysis of the Isolates Based on Inhibition ZoneDiameter Data All the Pseudomonas Aeromonas and het-erotrophic bacterial isolates were subjected to cluster analysisbased on their inhibition zone diameter (IZD) data anda dendrogram was generated using Wardrsquos method Thisapproach was used as a tool in determining the commonnessand in resolving differences between the MAR phenotypes ofdifferent isolates

Pseudomonas Aeromonas and heterotrophic bacteriaisolated from all sites were subjected to cluster analysisand two main clusters were observed (Figure 1) with thenumber of isolates from different sites within the clustersbeing depicted in Table 10 Both clusters have subclustersCluster 1 is a large cluster and has a total of 21 Aeromonasand heterotrophic bacterial isolates while cluster 2 is a mixed


Table 5 Percentage of total coliforms resistant to various antibiotics

Antibiotics KF AP C E OT K TM S A NE CIPMolopo eye N = 5 60 60 60 100 80 40 80 0 60 0 0Modimola dam N = 5 40 60 20 100 40 20 80 0 80 20 0Treated Molopo eye N = 1 100 100 100 100 100 100 100 0 100 0 0Treated dam N = 2 100 100 50 100 100 50 100 0 50 0 0Mixed water N = 2 100 100 100 100 50 50 100 0 100 0 0

Table 6 Percentage of faecal coliforms resistant to various antibiotics

Antibiotics KF AP C E OT K TM S A NE CIPMolopo eye N = 5 40 60 0 100 40 20 80 0 60 0 0Modimola dam N = 4 50 50 25 50 50 25 50 0 100 0 0

S2Tr

DH

1S5

EH1

S5M

PSS5

TrD

PSS5

DPS

S5EP

SS5

DA

ES5

EAE

S1Tr

EH1

S1D

H1

S3M

H1

S3Tr

EH1

S2D

H1

S2EH

1S5

TrH

1S5

DH

1S1

EH1

S3EH

1S4

TrEH

1S4

DA

ES4

EAE

S4EH

1S1

MH

1S1

TrD

H1

S4D

H1

S4Tr

DH

1S3

DH

1S3

DA

ES3

EAE0

50

100

150

200

250

300

Link

age d

istan

ce

Wardrsquos methodEuclidean distances

Figure 1 Cluster analysis for Pseudomonas Aeromonas and het-erotrophic bacteria isolated from different sites within the variousclusters (PS Pseudomonas AE Aeromonas H heterotrophic bacte-ria)

cluster with Pseudomonas Aeromonas and heterotrophicbacteria Pseudomonas is only found in cluster 2

The cluster analysis did not demonstrate groupings basedon bacterial groups or sites This suggests that all the isolateshad similar chemical exposure histories

35 Molecular Identification of Pseudomonas and AeromonasSpecies Specific PCR was used to determine the identities ofpresumptive Pseudomonas species through amplification ofthe gyrB toxA and the ecfX gene fragments Figures 2 and3 show agarose (1 wv) gels indicating gyrB and ecfX genefragments generated by PCR using genomic DNA extractedfrom Pseudomonas species isolated from different sites Gelelectrophoresis of PCR products revealed the desired 222 bpand 528 bp fragments for the gyrB and ecfX gene fragmentsrespectively A total of 61 isolates were screened and 17 werepositively identified

The identity of Aeromonas species was determined byscreening them for the presence of specific virulent genes

M 1 2 3 4 5 6 7 8 9

550bp

gyrB (222 bp)

Figure 2 Image of a composite agarose (1wv) gel depicting DNAextracted from Pseudomonas species Lane M (1 kb DNA Ladder)Lane 1ndash9 (gyrB gene fragments (222 bp) from Pseudomonas speciesisolated from different sites

M 1 2 3 4 5 6 7

ecfX (528bp)

Figure 3 Image of a composite agarose (1 wv) gel depictinggenomic DNA extracted from Pseudomonas species Lane M (1 kbDNA Ladder) Lane 1ndash7 ecfX gene fragments from Pseudomonasspecies isolated from different sites

aerolysin (aerA) [34] and haemolysin (hylH) [35] Figure 4shows agarose (1 wv) gels indicating hylH gene fragmentsgenerated by PCR using genomic DNA extracted fromAeromonas species isolated from different sites

PCR results depicted in Figures 2-4 support the biochem-ical identification of the Pseudomonas spp and Aeromonasspp Furthermore it demonstrated that certain virulencegenes were present in these isolates This is also supportedby positive 120573-haemolytic activity indicating the pathogenicpotential of these environmental isolated species


Table 7 Percentage of heterotrophic bacteria resistant to various antibiotics


Table 8 Percentage of Aeromonas spp resistant to various antibiotics

Antibiotics KF AP C E OT K TM S A NE CIPMolopo eye N = 4 25 25 25 100 50 0 50 0 75 0 0Modimola dam N = 2 50 100 50 100 100 50 100 50 100 50 0KF cephalothin AP ampicillin C chloramphenicol E erythromycin OT oxytetracycline K kanamycin TM trimethoprim S streptomycin A amoxicillinNE neomycin and CIP ciprofloxacin

Table 9 Prevalent antibiotic resistance phenotypes observedamongst the bacterial groups

Bacterial group Antibiotic resistance phenotypeFaecal coliform KF-AP-C-E-OT-K-TM-ATotal coliform KF-AP-C-E-OT-K-TM-AHeterotrophic bacteria KF-AP-C-E-OT-K-TM-AAeromonas sp KF-AP-C-E-OT-K-TM-S-A-NEPseudomonas sp KF-AP-C-E-OT-K-TM-AKF cephalothin AP ampicillin C chloramphenicol E erythromycinOT oxytetracycline K kanamycin TM trimethoprim S streptomycin Aamoxicillin and NE neomycin

Table 10 Number of heterotrophic bacteria (H) Pseudomonas (PS)and Aeromonas (AE) isolated from different sites within the variousclusters

SiteCluster 1

(Number of isolates = 21)Cluster 2

(Number of isolates = 8)H AE PS H AE PS

Dam 5 2 0 0 1 1Eye 4 2 0 1 1 1Treatedwater dam 2 0 0 1 0 1

Treatedwater eye 3 0 0 0 0 0

Mixedwater 2 0 0 0 0 1

4 Discussion

One of the objectives of this study was to isolate envi-ronmental bacteria from surface and drinking water inMafikeng North West Province South Africa A motiva-tion for this study was the numerous reports about theoccurrence of pathogenic microorganisms in drinking waterand the associated diseases [6 36ndash40] In addition to thisresistance of microorganisms to antibiotics of clinical inter-est has previously been reported in the area [24 26 29]The study demonstrated the occurrence of total coliforms

hylH 597bp

M 1 2 3 4

Figure 4 Image of a composite agarose (1 wv) gel depicting thehylH gene from Aeromonas species Lane M (1 kb DNA Ladder)Lanes 1ndash4 (hylH gene fragments from Aeromonas species isolatedfrom different sites)

faecal coliforms heterotrophic bacteria and Aeromonas andPseudomonas in water samples analysed which indicated theincidence of water contamination as some of these species areindicators of faecal contamination [8] These organisms mayharbour potential pathogens and the presence of pathogenicorganisms that can pose severe health risks to consumers ingeneral and immunocompromised individuals in particular[5] Reduction in the number of bacteria in the treated watercould be due to the treatment process when comparingdrinking water to raw water However occurrence of bacteriain the water after treatment could also harbour potentialpathogens and the health risk caused by these should betaken into consideration when water is distributed This is ofparticular importance when the drinking water abstractionand purification facility are at a relatively short distancefrom the sewage treatment and effluent disposal facility InMafikeng the latter is the case One of the waste watertreatment plants in Mafikeng is situated on the Modimoladam and is upstream from the drinking water purification


plant This may explain the larger number and diversity ofisolates from this water Results from this and other studiesshow that drinking water produced from the Modimola damshould be further analysed and that general microbiologicaltests that only include faecal coliforms and E coli may beinsufficient

Molopo eye is a protected spring into which no treatedsewage effluent is being dischargedHowever there is housingdevelopment that is linked to septic tanks The occurrence ofbacteria at this sitemay be due to contamination fromhumanactivity (septic tank diving) pets andwild birdsThe levels ofbacteria from this site were lower and after treatment very lownumbers of heterotrophic bacteria and no Aeromonas andPseudomonas spp were detected However the overall resultsobtained in the present study indicated that the organisms intreatedwatermay have survived the treatment processWhenthe levels ofPseudomonas spp in the rawwaterwere high thisincreased the chances of finding this species in treated water

A further objective of this study was to characterise theisolates using their antibiotic resistance profiles The resultsrevealed that a large proportion of the environmental isolateswere resistant to erythromycin followed by trimethoprimand amoxicillin The trend was in accordance with earlierstudies that showed resistance towards 120573-lactam macrolidesand phenicols [41 42] All the isolates from all the siteswere resistant to erythromycin which was in accordancewith the study conducted by [20] where most isolates werealso resistant to erythromycin and tetracycline (929) Alarge percentage (60ndash100) of isolates were also resistantto trimethoprim and ampicillin All these results could beattributed to the overuse of these antibiotics in the clinicaland veterinary setting All isolates were found to be sus-ceptible to streptomycin and ciprofloxacin in line with anearlier observation [43] This is not in agreement with yetanother observation [44] where increased resistance againstciprofloxacin in E coli and Pseudomonas was observed

Coliforms detected in all samples in the present studywere resistant to 3 or more classes of antibiotics This issupported by previous studies which obtained similar results[19 20] Multiple antibiotic resistance (MAR) in E coli andPseudomonas spp isolated from environmental samples andhospitalized patients had previously been reported [45ndash49]These studies support the findings of the present studyAntibiotic resistance patterns were generally similar in alldifferent sites However the largest numbers of isolates thatwere resistant to the largest numbers of antibiotics were fromthe Modimola dam

Although heterotrophic bacteria were isolated fromall sites and exhibited antibiotic resistance isolates fromall sites were susceptible to streptomycin neomycinand ciprofloxacin which agree with the susceptibility tociprofloxacin observed by Biyela et al [5] They found thehighest resistant towards trimethoprim followed by 120573-lactams and tetracycline This was also similar to the resultsby Biyela et al [5] All isolates from treated water fromMolopo eye and mixed water were sensitive to erythromycin

High loads of heterotrophic bacteria and opportunisticpathogens that harbour multiple drug resistance determi-nants pose significant health hazards to consumers especially

those whose immune systems are compromised [50] Asso-ciated virulence factors give heterotrophic bacteria the abil-ity to act as opportunistic pathogens [10] These bacteria(heterotrophic bacteria) are generally harmless but somemay harbour pathogenic features which may cause potentialhealth risks to humans and animalsThus the concern thatwereport here is the high levels of heterotrophic bacteria fromwater sources particularly drinking water from MafikengWhat is also of concern are the multiple antibiotic resistancepatterns observed amongst these isolatesThismay be a thera-peutic challenge if individuals infected by these opportunisticpathogens are to be treated by available antibiotics

In a study conducted by Sharma et al [7] all Aeromonasspp isolated from fresh water were resistant to ampicillinHowever in this study resistance to tetracycline trimetho-prim and cephalothinwas observed Furthermore resistanceof Aeromonas and Pseudomonas spp have also been reportedamong isolates from municipally treated tap and raw watersamples [51 52] These findings support the observations inthe present study that shows resistance of Aeromonas andPseudomonas spp to a variety of antibiotics

Wide distribution of antibiotic-resistant bacteria in sur-face and ground waters has been reported in previousstudies [53 54] and results of the present study is thus notuncommon There is however concern about the increasein the incidence of MAR in organisms from various sources[55] Resistance could be attributed to heavy contaminationfrom sewage effluent surface runoff agricultural activitieswild life industrial pollution and so forth Considering thehigh incidence of HIVAIDS in South Africa the importanceof such findings cannot be overemphasized The results ofthis study on bacterial resistance profiles are consistent withprevious studies in other surface and drinking water systems[10 20 55]

Besides receiving treated sewage effluent Modimola damalso supports small scale farming communities close to thedam Livestock graze around the dam and their faeces iswashed off into the dam during the rainy season Thereforethe dam could serve as a reservoir of antibiotic-resistantbacteria with sewage contamination contributing to thedissemination of antibiotic-resistant bacteria in this environ-ment There may thus be a link between the resistant isolatesfrom sewage treatment plantModimola dam and the treatedwater This aspect however needs to be demonstrated whichcould be done in follow-up studies Cluster analysis indicatedthat isolates from all sites had similar antibiotic resistanceprofiles This data indicates common chemical exposurehistories of the various isolates Such an approach could beused to investigate the relationship between sewage treatmentplant Modimola dam and treated waterThe results from thepresent study show that bacterial isolates from theModimoladam and treated water had similar antibiotic profiles

Another objective of this study was to identify thePseudomonas and Aeromonas species using PCR Isolateswere identified using gyrB ecfX and hylH The ecfX geneencodes an extra cytoplasmic function sigma factor whichmay be involved in haem uptake or virulence [56] whereasthe gyrB gene encodes the DNA gyrase subunit B a proteinwhich plays a crucial role in the DNA replication process


[34] and toxA encoding the exotoxin A precursor [33] PCRassay targeting the ecfX and gyrB genes is suitable for theidentification of P aeruginosa [56] In this study biochemicalidentification methods were supported by PCR assays toidentify the Pseudomonas spp isolated

Application of PCR technique to target gyrB aerA andhylH genes aremolecular markers for identifying and screen-ing potentially virulent Aeromonas species in food and theenvironment [34 35] In this study this approach supportedbiochemical data to identify the various Aeromonas spp Thedetection of Aeromonas species in the present study thatharbour putative virulence factors and resistance to variousantibiotics indicates that drinking water that is supplied tothe community could serve as a source for the transmissionof pathogens to humans [51 57]

Overall the results from the present highlight the needto conduct studies to determine the prevalence of antibiotic-resistant genes among environmental isolates and the dissem-ination of resistant genes among the pathogens This wouldprovide information about the health risks associated withthe consumption of contaminated water

5 Conclusions

An evaluation of the bacteriological quality of drinkingwater in the present study confirmed the presence of variousbacterial species including opportunistic pathogens such asAeromonas and Pseudomonas spp These organisms wereresistant to several classes of antibiotics Undesirable proper-ties of water quality caused by the presence of drug-resistantbacteria can pose a negative impact on human health

The data on multiple antibiotic resistance (MAR) profilesof bacterial isolates from water and the resistance patterns oforganisms in drinking water inMafikeng suggested that therehas been an indiscriminate use of the antibiotics tested Thehigh prevalence of multiple antibiotic-resistant organisms inthe drinking water distribution system could potentially posea threat to humans consuming this water The presence ofMAR organisms in the drinking water of Mafikeng SouthAfrica is an important health concern due to the risk of devel-oping waterborne diseases and the health risks associatedwith immunocompromised patients living in the area It istherefore imperative tomonitor the quality of water and strictquality control measures should be put in place to ensure theeffective treatment of drinking water Since the Modimoladam is the recipient of treated waste water as well as thewater source for drinking water production extremely strictmeasures should guide the waste water treatment plant Thequality of effluent leaving in this plant should be extremelyhigh This would decrease the load of microorganisms (andother contaminants) allowed to enter the dam Suchmeasureswill improve the overall quality of water available for drinkingwater production preventing outbreaks and spreading waterborne diseases Antibiotic resistance surveillance can be usedas tool to control the problem of antibiotic resistance andto educate the public on the consequences of the misuse ofantibiotics and also to regulate the usage of drugs in bothhuman and veterinarymedicine It is also helpful to formulate

guidelines for the optimal use of antibiotics Further studiesshould be conducted to assess the level of antibiotics in waterand the potential risks associated with human consumptionof polluted water It is also very important that findings fromstudies such as this one should be disseminated to the relevantstakeholders and the affected communities

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank the National ResearchFoundation (FA20040101000030 and FA2006040700029) forthe financial support of this study The assistance receivedfrom Mrs Rika Huyser and the employees of MmabathoWater Works is also hereby acknowledged

References

[1] C Faria I Vaz-Moreira E Serapicos O C Nunes and C MManaia ldquoAntibiotic resistance in coagulase negative staphylo-cocci isolated from wastewater and drinking waterrdquo Science ofthe Total Environment vol 407 no 12 pp 3876ndash3882 2009

[2] WHO Guidelines for Drinking Water Quality vol 1 WorldHealth Organization Geneva Switzerland 3rd edition 2004

[3] J Fawell and M J Nieuwenhuijsen ldquoContaminants in drinkingwater Environmental pollution and healthrdquo British MedicalBulletin vol 68 no 1 pp 199ndash208 2003

[4] S Volker C Schreiber and T Kistemann ldquoDrinking waterquality in household supply infrastructuremdasha survey of thecurrent situation in Germanyrdquo International Journal of Hygieneand Environmental Health vol 213 no 3 pp 204ndash209 2010

[5] P T Biyela J Lin and C C Bezuidenhout ldquoThe role of aquaticecosystems as reservoirs of antibiotic resistant bacteria andantibiotic resistance genesrdquo Water Science and Technology vol50 no 1 pp 45ndash50 2004

[6] C L Obi N Potgieter P O Bessong and G MatsaungldquoAssessment of the microbial quality of river water sources inrural Venda communities in South Africardquo Water SA vol 28no 3 pp 287ndash292 2002

[7] A Sharma N Dubey and B Sharan ldquoCharacterization ofaeromonads isolated from the river Narmada Indiardquo Interna-tional Journal of Hygiene and Environmental Health vol 208no 5 pp 425ndash433 2005

[8] L F Webster B C Thompson M H Fulton et al ldquoIdentifica-tion of sources of Escherichia coli in South Carolina estuariesusing antibiotic resistance analysisrdquo Journal of ExperimentalMarine Biology and Ecology vol 298 no 2 pp 179ndash195 2004

[9] S Dumontet K Krovacek S B Svenson V Pasquale SB Baloda and G Figliuolo ldquoPrevalence and diversity ofAeromonas and Vibrio spp in coastal waters of Southern ItalyrdquoComparative Immunology Microbiology and Infectious Diseasesvol 23 no 1 pp 53ndash72 2000

[10] D Pavlov C M E deWet W O K Grabow and M M EhlersldquoPotentially pathogenic features of heterotrophic plate countbacteria isolated from treated and untreated drinking waterrdquoInternational Journal of Food Microbiology vol 92 no 3 pp275ndash287 2004


[11] R Eberhard and P Robinson Guidelines for the Development ofNational Water Policies and Strategies to Support IWRM 2003Draft SADC Water Sector Co-Ordination Unit GaboroneBotswana 2003

[12] J R Adewumi A A Ilemobade and J E Van Zyl ldquoTreatedwastewater reuse in South Africa overview potential andchallengesrdquo Resources Conservation and Recycling vol 55 no2 pp 221ndash231 2010

[13] International Water Association (IWA) 2008[14] R Mckenzie J Buckle W Wegelin and N Meyer ldquoWater

demand management cookbook rand water in collaborationwith WRP managing water for African Cities UN HabitatProgramme New Yorkrdquo Medical Bulletin vol 68 pp 199ndash2082003

[15] S Dimitriadis Issues Encountered in Advancing AustraliarsquosWater Recycling Schemes Research Brief Parliamentary LibraryParliament of Australia Department of Parliamentary Services2005

[16] B Vaseeharan P Ramasamy T Murugan and J C ChenldquoIn vitro susceptibility of antibiotics against Vibrio spp andAeromonas spp isolated from Penaeus monodon hatcheries andpondsrdquo International Journal of Antimicrobial Agents vol 26no 4 pp 285ndash291 2005

[17] J L Martinez ldquoEnvironmental pollution by antibiotics andby antibiotic resistance determinantsrdquo Environmental Pollutionvol 157 no 11 pp 2893ndash2902 2009

[18] M Akram M Shahid and A U Khan ldquoEtiology and antibioticresistance patterns of community-acquired urinary tract infec-tions in J N M C Hospital Aligarh Indiardquo Annals of ClinicalMicrobiology and Antimicrobials vol 6 article 4 pp 1ndash10 2007

[19] A Łuczkiewicz K Jankowska S Fudala-Ksiazek and KOlanczuk-Neyman ldquoAntimicrobial resistance of fecal indica-tors in municipal wastewater treatment plantrdquoWater Researchvol 44 no 17 pp 5089ndash5097 2010

[20] J E Moore P J A Moore B C Millar et al ldquoThe presence ofantibiotic resistant bacteria along the River LaganrdquoAgriculturalWater Management vol 98 no 1 pp 217ndash221 2010

[21] T Schwartz W Kohnen B Jansen and U Obst ldquoDetectionof antibiotic-resistant bacteria and their resistance genes inwastewater surface water and drinking water biofilmsrdquo FEMSMicrobiology Ecology vol 43 no 3 pp 325ndash335 2003

[22] T I Lobova Y V Barkhatov O V Salamatina and L Y PopovaldquoMultiple antibiotic resistance of heterotrophic bacteria in thelittoral zone of Lake Shira as an indicator of human impact onthe ecosystemrdquoMicrobiological Research vol 163 no 2 pp 152ndash160 2008

[23] Y Zhang C F Marrs C Simon and C Xi ldquoWastewater treat-ment contributes to selective increase of antibiotic resistanceamongAcinetobacter spprdquo Science of the Total Environment vol407 no 12 pp 3702ndash3706 2009

[24] S G Mulamattathil H A Esterhuysen and P J Preto-rius ldquoAntibiotic-resistant Gram-negative bacteria in a virtuallyclosed water reticulation systemrdquo Journal of Applied Microbiol-ogy vol 88 no 6 pp 930ndash937 2000

[25] J Lin and P T Biyela ldquoConvergent acquisition of antibioticresistance determinants amongst the Enterobacteriaceae iso-lates of the Mhlathuze River KwaZulu-Natal (RSA)rdquoWater SAvol 31 no 2 pp 257ndash260 2005

[26] C W Kinge and M Mbewe ldquoCharacterisation of Shigellaspecies isolated from river catchments in the North Westprovince of South Africardquo South African Journal of Science vol106 no 11-12 article 211 2010

[27] SANS South African National Standards ldquoDrinking watermdashpart 1 microbiological physical chemical aesthetic andchemical determinandsrdquo SABS Standards Division PretoriaSouth Africa 2011

[28] W Kirby W Bauer J Sherris and M Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoTheAmerican Journal of Clinical Pathology vol 45 no 4 pp 493ndash496 1966

[29] C N AtebaMMbewe andC C Bezuidenhout ldquoPrevalence ofEscherichia coliO157 strains in cattle pigs and humans in NorthWest province South Africardquo South African Journal of Sciencevol 104 no 1-2 pp 7ndash8 2008

[30] National Committee for Clinical Laboratory Standards ldquoPer-formance standards for antimicrobial disk and dilution sus-ceptibility tests for bacterial isolates from animalsrdquo NationalCommittee for Clinical Laboratory StandardsWayne Pa USAApproved standard M31-A19 (11) 1999

[31] X Qin J Emerson J Stapp L Stapp P Abe and J L BurnsldquoUse of real-time PCR with multiple targets to identify Pseu-domonas aeruginosa and other nonfermenting gram-negativebacilli from patients with cystic fibrosisrdquo Journal of ClinicalMicrobiology vol 41 no 9 pp 4312ndash4317 2003

[32] A A Khan and C E Cerniglia ldquoDetection of Pseudomonasaeruginosa from clinical and environmental samples by ampli-fication of the exotoxin A gene using PCRrdquo Applied andEnvironmentalMicrobiology vol 60 no 10 pp 3739ndash3745 1994

[33] R Lavenir D Jocktane F Laurent S Nazaret and BCournoyer ldquoImproved reliability of Pseudomonas aeruginosaPCR detection by the use of the species-specific ecfX genetargetrdquo Journal of Microbiological Methods vol 70 no 1 pp 20ndash29 2007

[34] E Kaszab S Szoboszlay C Dobolyi J Hahn N Pek andB Kriszt ldquoAntibiotic resistance profiles and virulence markersof Pseudomonas aeruginosa strains isolated from compostsrdquoBioresource Technology vol 102 no 2 pp 1543ndash1548 2011

[35] A H Yousr S Napis G R A Rusul and R Son ldquoDetectionof aerolysin and hemolysin genes in Aeromonas spp isolatedfrom environmental and shellfish sources by polymerase chainreactionrdquo ASEAN Food Journal vol 14 no 2 pp 115ndash122 2007

[36] A I Schafer H M A Rossiter P A Owusu B S Richards andE Awuah ldquoPhysico-chemical water quality in Ghana prospectsfor water supply technology implementationrdquoDesalination vol248 no 1ndash3 pp 193ndash203 2009

[37] P T Yillia N Kreuzinger and J MMathooko ldquoThe effect of in-stream activities on the Njoro River Kenya Part I stream flowand chemical water qualityrdquo Physics and Chemistry of the Earthvol 33 no 8ndash13 pp 722ndash728 2008

[38] H Schraft and L A Watterworth ldquoEnumeration of het-erotrophs fecal coliforms and Escherichia coli in water compar-ison of 3M Petrifilm plates with standard plating proceduresrdquoJournal of Microbiological Methods vol 60 no 3 pp 335ndash3422005

[39] W Germs M S Coetzee L van Rensburg and M S MaboetaldquoA preliminary assessment of the chemical and microbial waterquality of the Chunies RivermdashLimpopordquoWater SA vol 30 no2 pp 267ndash272 2004

[40] J T Carter E W Rice S G Buchberger and Y Lee ldquoRela-tionships between levels of heterotrophic bacteria and waterquality parameters in a drinking water distribution systemrdquoWater Research vol 34 no 5 pp 1495ndash1502 2000

[41] C L Obi P O BessongMMomba N Potgieter A Samie andE O Igumbor ldquoProfiles of antibiotic susceptibilities of bacterial


isolates and physico-chemical quality of water supply in ruralVenda communities South AfricardquoWater SA vol 30 no 4 pp515ndash519 2004

[42] P Messi E Guerrieri and M Bondi ldquoAntibiotic resistance andantibacterial activity in heterotrophic bacteria of mineral wateroriginrdquo Science of the Total Environment vol 346 no 1ndash3 pp213ndash219 2005

[43] J Lin P T Biyela and T Puckree ldquoAntibiotic resistance profilesof environmental isolates from Mhlathuze River KwaZulu-Natal (RSA)rdquoWater SA vol 30 no 1 pp 23ndash28 2004

[44] G J Oudhuis A Verbon J A A Hoogkamp-Korstanje andE E Stobberingh ldquoAntimicrobial resistance in Escherichia coliand Pseudomonas aeruginosa from Intensive Care Units in TheNetherlands 1998ndash2005rdquo International Journal of AntimicrobialAgents vol 31 no 1 pp 58ndash63 2008

[45] T C Siewicki T Pullaro W Pan S McDaniel R Glenn and JStewart ldquoModels of total and presumed wildlife sources of fecalcoliform bacteria in coastal pondsrdquo Journal of EnvironmentalManagement vol 82 no 1 pp 120ndash132 2007

[46] A Sorlozano J Gutierrez J Luna et al ldquoHigh presence ofextended-spectrum 120573-lactamases and resistance to quinolonesin clinical isolates of Escherichia colirdquoMicrobiological Researchvol 162 no 4 pp 347ndash354 2007

[47] M E Scott A R Melton-Celsa and A D OrsquoBrien ldquoMutationsin hns reduce the adherence of Shiga toxin-producing E coli091H21 strain B2F1 to human colonic epithelial cells andincrease the production of hemolysinrdquo Microbial Pathogenesisvol 34 no 3 pp 155ndash159 2003

[48] E Tumeo H Gbaguidi-Haore I Patry X Bertrand MThouverez andD Talon ldquoAre antibiotic-resistant Pseudomonasaeruginosa isolated from hospitalised patients recovered inthe hospital effluentsrdquo International Journal of Hygiene andEnvironmental Health vol 211 no 1-2 pp 200ndash204 2008

[49] E Breidenstein C de la Fuente-Nunez and R HancockldquoPseudomonas aeruginosa all roads lead to resistancerdquo Trendsin Microbiology vol 19 no 8 pp 419ndash426 2011

[50] M I Jeena P Deepa KMMujeeb Rahiman R T Shanthi andAAMHatha ldquoRisk assessment of heterotrophic bacteria frombottled drinking water sold in Indian marketsrdquo InternationalJournal of Hygiene and Environmental Health vol 209 no 2pp 191ndash196 2006

[51] G Emekdas G Aslan S Tezcan et al ldquoDetection of thefrequency antimicrobial susceptibility and genotypic discrim-ination of Aeromonas strains isolated from municipally treatedtap water samples by cultivation and AP-PCRrdquo InternationalJournal of Food Microbiology vol 107 no 3 pp 310ndash314 2006

[52] E E Odjadjare E O Igbinosa R Mordi B Igere C LIgeleke and A I Okoh ldquoPrevalence of multiple antibioticsresistant (MAR) Pseudomonas species in the final effluentsof three municipal wastewater treatment facilities in SouthAfricardquo International Journal of Environmental Research andPublic Health vol 9 no 6 pp 2092ndash2107 2012

[53] S Harakeh H Yassine and E Mutasem ldquoAntimicrobial-resistance patterns of Escherichia coli and Salmonella strainsin the aquatic Lebanese environmentsrdquo Environment Pollutionvol 295 no 2 pp 503ndash511 2005

[54] KKummerer ldquoAntibiotic in the aquatic environment A reviewpart 2rdquo Chemosphere vol 75 pp 435ndash441 2009

[55] R Shrivastava R K Upreti S R Jain K N Prasad P KSeth and U C Chaturvedi ldquoSuboptimal chlorine treatmentof drinking water leads to selection of multidrug-resistant

Pseudomonas aeruginosardquo Ecotoxicology and EnvironmentalSafety vol 58 no 2 pp 277ndash283 2004

[56] S N Anuj D M Whiley T J Kidd et al ldquoIdentification ofPseudomonas aeruginosa by a duplex real-time polymerasechain reaction assay targeting the ecfX and the gyrB genesrdquoDiagnosticMicrobiology and Infectious Disease vol 63 no 2 pp127ndash131 2009

[57] X Li M Mehrotra S Ghimire and L Adewoye ldquo120573-Lactamresistance and 120573-lactamases in bacteria of animal originrdquoVeterinary Microbiology vol 121 no 3-4 pp 197ndash214 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Africa and the world at large waste water reuse can forman important component of water demandmanagement [13]but this waste water reuse may affect the quality of drinkingwater if proper treatment procedures are not implementedWhereas waste water reuse has been extensively implementedin some European and African countries [13] yet in SouthAfrica only a few waste water reuse schemes have beendocumented [14 15] and there is limited implementation ofthis alternative in communities

Alarming increases in the consumption of antibioticsthrough human therapy and agricultural processes have beenreported [16 17] and this extensive usage in both humanand animal medicine has resulted in the development ofantibiotic-resistant bacteria which affect the treatment ofinfections [18 19] Antibiotic resistance has therefore becomea major public health issue [20] and its presence in wastewater surface water and drinking water is well documented[19ndash22] The hazard associated with the pathogenicity ofmicrobes is aggravated by its ability to resist destructionby antibiotics Biological treatment processes in the wastewater treatment plants may result in a selective increaseof antibiotic-resistant bacteria and therefore increase theoccurrence of multidrug-resistant organisms [23] Althoughmicroorganisms in drinking water are reduced by chlorina-tion they may survive the treatment process and enter thedistribution system [1] Moreover the presence of antibioticresistance in microorganisms has been previously reported[24ndash26] Considering the fact that the public health of acommunity may be related to the quality of treated wastewater supplied and that public health can be protected byreducing the pathogenic microorganisms in drinking waterthe present study was designed to isolate environmentalbacteria from surface and drinking water in Mafikeng andidentify the Pseudomonas and Aeromonas species usingpolymerase chain reaction (PCR) A further objective wasto characterize the isolates using their antibiotic resistanceprofiles

2 Materials and Methods

21 Study Area Water samples were collected from fivesampling points around Mafikeng namely both raw andtreated (drinking) water from a karstic groundwater sourcethe Molopo Eye both raw water and treated (drinking)water from the Modimola dam and finally mixed watertreated water from both sources mixed in the Signal hillreservoir and distributed to some areas in the city Thesesampling pointswere chosen for the study becausewater fromMolopo eye andModimola dam after purification is used forhuman consumption and for recreational agricultural andindustrial purposes As few small scale of farmers live nearthese two water resources and the Modimola Dam receivestreated sewage effluent from theMmabatho sewage treatmentplant which is the major source of pathogens it is thereforeimportant to investigate the microbiological quality of waterat these points

22 Sampling Raw and treated water samples were collectedduring a one-year period in February April July and

October to cover the four different seasons Water samplesfrom the Molopo eye and Modimola dam were collectedaseptically in sterile 500mL Duran Schott glass bottles fromdifferent sampling points by directly dipping the bottles intothe surface of the water Purifiedwater samples were collecteddirectly in to the sterile bottles from the tap after letting thetap run for a minute The samples were labelled properly andtransported on ice to the laboratory for analysis Aliquotsof the samples were used for selective isolation of faecalcoliforms total coliforms Aeromonas Pseudomonas andheterotrophic bacteria based on standard microbiologicalprocedures [27]

23 Isolation Purification and Characterization ofPlanktonic Bacteria

231 Isolation by Membrane Filtration For all the samplesthree volumes of 100mL were filtered through 045120583m pore-sized filter (cellulose nitrate membranes Whatman Labo-ratory Division Maidstone England) using a water pump(model Sartorius 16824) These membranes were asepticallyplaced up on plates with appropriate selectivemedia ensuringthat no air bubbles were trapped The selective media usedare as follows mFC agar used as a selective medium forfaecal coliforms mEndo for total coliforms nutrient agar forheterotrophic bacteria and Aeromonas selective medium forAeromonas and Pseudomonas (Biolab Merck South Africa)All the media were prepared according to the manufacturersrsquoinstructions (Biolab Merck South Africa) Each samplewas analysed in triplicate In order to isolate heterotrophicbacteria 1mL of the treated water samples was spread ontothe nutrient agar plates Water samples from Modimola damand Molopo eye were serially diluted and 1mL of the 5 foldserial dilutions was spread on to the nutrient agar plates

The plates were incubated at 37∘C except for mFC agarwhich were incubated at 45∘C for 24 hours The colonieswere enumerated characterized and recorded The resultswere expressed as the number of faecal coliforms total col-iforms Pseudomonas and Aeromonas in 100mL of water andheterotrophic bacteria in 1mL of water Blue colonies frommFC agar (presumptive coliforms) metallic-sheen coloniesfrom mEndo agar (presumptive total coliforms) and yellow(presumptive Aeromonas) and green colonies (presumptivePseudomonas) from Aeromonas selective media were pickedfor further work (Biolab Catalogue)

232 Purification of Colonies Colonies were purified bytwice subculturing using the streaking plate method Youngcultures were used for Gram staining and all isolates wereidentified as Gram-negative Bacilli All the Gram-negativeisolates were subjected to primary and secondary biochemi-cal identification testsThe bacteria that were picked to createantibiogramswere streaked on to nutrient agar slants tomakesample cultures and for PCR purpose

24 Antimicrobial Susceptibility Testing An antibiotic sus-ceptibility test was performed using the Kirby-Bauer diskdiffusion method [28] The following antibiotic discs (Mast








2S this














ECF1ECF2



GyrPA-398GyrPA-620



ETA1ETA2













3 Results



















MD treated water





Mixed water













S2Tr

DH

1S5

EH1

S5M

PSS5

TrD

PSS5

DPS

S5EP

SS5

DA

ES5

EAE

S1Tr

EH1

S1D

H1

S3M

H1

S3Tr

EH1

S2D

H1

S2EH

1S5

TrH

1S5

DH

1S1

EH1

S3EH

1S4

TrEH

1S4

DA

ES4

EAE

S4EH

1S1

MH

1S1

TrD

H1

S4D

H1

S4Tr

DH

1S3

DH

1S3

DA

ES3

EAE0

50

100

150

200

250

300

Link

age d

istan

ce







M 1 2 3 4 5 6 7 8 9

550bp

gyrB (222 bp)


M 1 2 3 4 5 6 7

ecfX (528bp)












SiteCluster 1






4 Discussion


hylH 597bp

M 1 2 3 4



















5 Conclusions






Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























2S this














ECF1ECF2



GyrPA-398GyrPA-620



ETA1ETA2













3 Results



















MD treated water





Mixed water













S2Tr

DH

1S5

EH1

S5M

PSS5

TrD

PSS5

DPS

S5EP

SS5

DA

ES5

EAE

S1Tr

EH1

S1D

H1

S3M

H1

S3Tr

EH1

S2D

H1

S2EH

1S5

TrH

1S5

DH

1S1

EH1

S3EH

1S4

TrEH

1S4

DA

ES4

EAE

S4EH

1S1

MH

1S1

TrD

H1

S4D

H1

S4Tr

DH

1S3

DH

1S3

DA

ES3

EAE0

50

100

150

200

250

300

Link

age d

istan

ce







M 1 2 3 4 5 6 7 8 9

550bp

gyrB (222 bp)


M 1 2 3 4 5 6 7

ecfX (528bp)












SiteCluster 1






4 Discussion


hylH 597bp

M 1 2 3 4



















5 Conclusions






Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















ECF1ECF2



GyrPA-398GyrPA-620



ETA1ETA2













3 Results



















MD treated water





Mixed water













S2Tr

DH

1S5

EH1

S5M

PSS5

TrD

PSS5

DPS

S5EP

SS5

DA

ES5

EAE

S1Tr

EH1

S1D

H1

S3M

H1

S3Tr

EH1

S2D

H1

S2EH

1S5

TrH

1S5

DH

1S1

EH1

S3EH

1S4

TrEH

1S4

DA

ES4

EAE

S4EH

1S1

MH

1S1

TrD

H1

S4D

H1

S4Tr

DH

1S3

DH

1S3

DA

ES3

EAE0

50

100

150

200

250

300

Link

age d

istan

ce







M 1 2 3 4 5 6 7 8 9

550bp

gyrB (222 bp)


M 1 2 3 4 5 6 7

ecfX (528bp)












SiteCluster 1






4 Discussion


hylH 597bp

M 1 2 3 4



















5 Conclusions






Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























MD treated water





Mixed water













S2Tr

DH

1S5

EH1

S5M

PSS5

TrD

PSS5

DPS

S5EP

SS5

DA

ES5

EAE

S1Tr

EH1

S1D

H1

S3M

H1

S3Tr

EH1

S2D

H1

S2EH

1S5

TrH

1S5

DH

1S1

EH1

S3EH

1S4

TrEH

1S4

DA

ES4

EAE

S4EH

1S1

MH

1S1

TrD

H1

S4D

H1

S4Tr

DH

1S3

DH

1S3

DA

ES3

EAE0

50

100

150

200

250

300

Link

age d

istan

ce







M 1 2 3 4 5 6 7 8 9

550bp

gyrB (222 bp)


M 1 2 3 4 5 6 7

ecfX (528bp)












SiteCluster 1






4 Discussion


hylH 597bp

M 1 2 3 4



















5 Conclusions






Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















S2Tr

DH

1S5

EH1

S5M

PSS5

TrD

PSS5

DPS

S5EP

SS5

DA

ES5

EAE

S1Tr

EH1

S1D

H1

S3M

H1

S3Tr

EH1

S2D

H1

S2EH

1S5

TrH

1S5

DH

1S1

EH1

S3EH

1S4

TrEH

1S4

DA

ES4

EAE

S4EH

1S1

MH

1S1

TrD

H1

S4D

H1

S4Tr

DH

1S3

DH

1S3

DA

ES3

EAE0

50

100

150

200

250

300

Link

age d

istan

ce







M 1 2 3 4 5 6 7 8 9

550bp

gyrB (222 bp)


M 1 2 3 4 5 6 7

ecfX (528bp)












SiteCluster 1






4 Discussion


hylH 597bp

M 1 2 3 4



















5 Conclusions






Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























SiteCluster 1






4 Discussion


hylH 597bp

M 1 2 3 4



















5 Conclusions






Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































5 Conclusions






Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article isolation of environmental bacteria from surface...

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