research article detection of extended spectrum beta

Research ArticleDetection of Extended Spectrum Beta-Lactamases ResistanceGenes among Bacteria Isolated from Selected Drinking WaterDistribution Channels in Southwestern Nigeria

Ayodele T Adesoji1 and Adeniyi A Ogunjobi2

1Department of Biological Sciences Federal University Dutsin-Ma Katsina Road PMB 5001 Dutsin-Ma Katsina State Nigeria2Department of Microbiology University of Ibadan Oyo State Nigeria

Correspondence should be addressed to Ayodele T Adesoji timmyayus2002yahoocom

Received 11 April 2016 Accepted 4 July 2016

Academic Editor Carla R Arciola

Copyright copy 2016 A T Adesoji and A A Ogunjobi 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

Extended Spectrum Beta-Lactamases (ESBL) provide high level resistance to beta-lactam antibiotics among bacteria In thisstudy previously described multidrug resistant bacteria from raw treated and municipal taps of DWDS from selected dams insouthwestern Nigeria were assessed for the presence of ESBL resistance genes which include 119887119897119886TEM 119887119897119886SHV and 119887119897119886CTX by PCRamplification A total of 164 bacteria spread across treated (33) raw (66) and municipal taps (68) belonging to 120572-Proteobacteria120573-Proteobacteria 120574-Proteobacteria Flavobacteriia Bacilli and Actinobacteria group were selected for this study Among thesebacteria the most commonly observed resistance was for ampicillin and amoxicillinclavulanic acid (61 isolates) Sixty-one isolatescarried at least one of the targeted ESBL genes with 119887119897119886TEM being the most abundant (5061) and 119887119897119886CTX being detected least(361) Klebsiella was the most frequently identified genus (1803) to harbour ESBL gene followed by Proteus (1475) Moreovercombinations of two ESBL genes 119887119897119886SHV + 119887119897119886TEM or 119887119897119886CTX + 119887119897119886TEM were observed in 11 and 1 isolate respectively In conclusionclassic 119887119897119886TEM ESBL gene was present in multiple bacterial strains that were isolated from DWDS sources in Nigeria Theseenvironments may serve as foci exchange of genetic traits in a diversity of Gram-negative bacteria

1 Introduction

Access to safe drinkingwater is essential for human health [1]While access to safe and affordable water should be availableto everyone this remains a challenge in low- and middle-income countries including Nigeria which is the mostpopulous country in Africa Safe drinking water is mostlyviewed in terms of organic and inorganic contaminants butalso in terms of biological contamination In this respect lessattention has been given to the role that water may play inthe dissemination of antibiotic resistance traits in populationsthat are exposed to substandard water on a daily basis [2ndash5]

Arguably the most clinically important antibiotic resis-tance genes are those that encode enzymes that hydrolyze 120573-lactams (bla genes) [6] These traits confer high level resis-tance to 120573-lactam antibiotics which are the most widely used

antibiotics in clinical and veterinary practice [7 8] ExtendedSpectrum 120573-Lactamase (ESBL) is group of enzymes that canhydrolyze a variety of 120573-lactams including cephalosporinslike ceftazidime cefotaxime and ceftriaxone and monobac-tams like aztreonam in addition to penicillin but does nothydrolyze cephamycins like cefoxitin Most of the ESBL alsohave the ability to hydrolyze fourth-generation cephalospo-rins including cefepime [9]

A variety of transferable genes encoding 120573-lactamaseactivity have been described in clinical environments includ-ing 119887119897119886CTX-M 119887119897119886GES 119887119897119886HER 119887119897119886OXA 119887119897119886OXY 119887119897119886SED 119887119897119886SHV119887119897119886SPM 119887119897119886VEB 119887119897119886VIM and ampC [10] Among themost com-mon bla genes is the 119887119897119886TEM-1 gene the first described bla geneand a representative of the 119887119897119886TEM group that now consistsof more than 220 different distinct variants (ldquoallelesrdquo) whichencode different amino acid polymorphisms that extend their

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 7149295 9 pageshttpdxdoiorg10115520167149295

2 BioMed Research International

substrate range (httpwwwlaheyorgStudiestemtableasp)[10]

Previous reports indicated that multidrug resistant bac-teria are present in drinking water distribution systemsfrom southwestern Nigeria [3ndash5] These bacteria encodedresistance to a diversity of beta-lactams including ceftiofurampicillin and combination of amoxicillin and amoxicillinclavulanic acid

The aim of this study was to genotype MDR bacteriaisolated from our previous studies [3ndash5] for the presence ofselected beta-lactamase resistance genes using PCR

2 Materials and Methods

21 Dam Description Sampling Selection Isolation Storageand Molecular Characterization of Bacteria The descriptionof sampled dams in this study is in our previous publications[3ndash5] Moreover for clarity of this paper ninety-six watersamples were purposively collected aseptically into sterilescrew cap bottles from six selected water distribution sys-tems of dams in Ife Ede Asejire Eleyele Owena Ondoand Owena-Idanre in southwestern Nigeria Samples werecollected four times between December 2010 and July 2011from raw treated and two randomly selected municipaldistribution taps Afterwards samples were serially dilutedand plated on Nutrient agar eosin methylene blue agar(EMB) and Deoxycholate agar (DCA) Thereafter bacteriawere picked with the aim of maximizing the diversity ofcolony morphology represented from each sample Pickedcolonies were restreaked on Nutrient agar to obtain purecultures These were subsequently transferred to Nutrientagar slants and also stored in phosphate buffer glycerol atminus80∘C [3ndash5]Molecular characterization of bacteria using 16SrDNA sequencing was determined as described in Adesoji etal [11]

22 Antibiotic Susceptibility Testing Agar dilution assays(also called breakpoint assays) were conducted using Luria-Bertani agar with seeded antibiotics used to assess antibioticsusceptibility Antibiotics concentrations used for Gram-negative bacteria included florfenicol (16120583gmL) tetracy-cline (16 120583gmL) streptomycin (16 120583gmL) gentamycin(16 120583gmL) kanamycin (64 120583gmL) chloramphenicol(32 120583gmL) nalidixic acid (30 120583gmL) amoxicillinclavulanicacid (3216 120583gmL) ceftiofur (12120583gmL) sulfamethoxazole(512120583gmL) and sulfamethoxazoletrimethoprim (764 120583gmL) Antibiotics concentrations used for Gram-positivebacteria include sulfamethoxazole (512120583gmL) ampicillin(05 120583gmL) tetracycline (16 120583gmL) sulfamethoxazoletri-methoprim (764120583gmL) gentamycin (16 120583gmL) erythro-mycin (8120583gmL) rifampin (4120583gmL) lincomycin (4120583gmL)and ciprofloxacin (4 120583gmL) Negative and positive controlsused were E coli strain K12 and E coli strain H4H respec-tively as we described in our previous studies [3ndash5 11]

23 Resistance Genotyping PCR testing was conducted forbacteria having resistance to ge3 classes of antibiotics includ-ing resistance to amoxicillinclavulanic acid ceftiofur orampicillinThereafter forward and reverse primer specific for

selected ESBL genes included 119887119897119886SHV (SHV F 51015840-GCGAAA-GCCAGCTGTCGGGC-31015840 and SHV R 51015840-GATTGGCGG-CGCTGTTATCGC-3) 119887119897119886CTX M (CTX F 51015840-GTGCAG-TACCAGTAAAGTTATGG-31015840 and CTX R 51015840-CGCAAT-ATCATTGGTGGTGCC-31015840) and 119887119897119886TEM (TEM F 51015840-AAA-GATGCTGAAGATCA-31015840 and TEM R 51015840-TTTGGTATG-GCTTCATTC-31015840) [12] Condition for 119887119897119886SHV PCR included1min denaturation (95∘C followed by 30 cycles of 96∘C for30 s 62∘C for 30 s and 72∘C for 30 s and final extension of72∘C for 10min Conditions were identical for other assaysexcept the annealing temperatures which were 55∘C and44∘C for 119887119897119886CTX-M and 119887119897119886TEM respectively Afterwards PCRproducts were separated sized and visualized by using 1agarose gel electrophoresis to confirm amplification

3 Results

31 Bacteria Isolates Isolates used in this study wereselected from our previous studies [3ndash5] and represented120572-Proteobacteria 120573-Proteobacteria 120574-ProteobacteriaFlavobacteriia Bacilli and Actinobacteria with 33 66 and68 being isolated from all treated raw and municipal tapsrespectively (Table 1) Proteus was the most frequent (1818)isolated Gram-negative genus from the treated water whileKlebsiella was the most frequently (1515) isolated genusfrom raw water Bacillus was the most common isolatedGram-positive genus for treated and municipal water

32 PCR-Positive Isolates In this study 61 isolates out of164 MDR isolates were PCR-positive for at least one tar-geted gene Highest occurrence of bla gene among Gram-negative bacteria compared to Gram-positive bacteria wasobserved Most commonly isolated genus carrying bla geneis Klebsiella (1803) followed by Proteus spp (1475)119861119897119886TEM was detected in the majority of beta-lactam resis-tant isolates (5061) while 119887119897119886CTX was rarely detected (361)(Table 2) A combination of two genes 119887119897119886SHV + 119887119897119886TEMor 119887119897119886CTX + 119887119897119886TEM was observed in 11 and 1 bacteriarespectively (Table 2) Other genera including AquitaleaComamonas Enterobacter Leucobacter Lysinibacillus Pan-toea Pseudochrobactrum Sphingobacterium and Ralstoniawere tested but were PCR-negative for resistance genes

4 Discussion

The hazard associated with the pathogenicity of microbes isaggravated by its ability to resist destruction by antibiotics [2]In this study beta-lactamase producing bacteria and genes(ie 119887119897119886CTX and 119887119897119886TEM) were detected from every sampledwater distribution system This is similar to the report of Xiet al [13] who also investigated the prevalence and dynamicsof heterotrophic antibiotics resistance bacteria and genesin drinking water source and treated drinking water usingculture-dependent methods and molecular techniques Theauthors observed the presence of 119887119897119886TEM and 119887119897119886SHV genesin all water samples except one which is evidence that thesegenes are distributed widely in drinking water systems Thisis similar to what we also reported in Table 3 showing thespread of these beta-lactamase resistance genes among all

BioMed Research International 3

Table 1 Classes and families of selected bacteria

Source Class Family Number ( of total from source)

Raw water

120572-Proteobacteria Brucellaceae 1 (151)

120573-Proteobacteria Alcaligenaceae 9 (1364)Neisseriaceae 1 (151)

120574-Proteobacteria

Enterobacteriaceae 27 (4091)Moraxellaceae 2 (303)

Aeromonadaceae 6 (909)Xanthomonadaceae 2 (303)

Flavobacteriia Myroidaceae 1 (151)Uncultured bacteria clone 3 (455)

Bacilli Bacillaceae 10 (1515)Staphylococcaceae 3 (455)

Actinobacteria Microbacteriaceae 1 (151)Total raw water 66

Treated water

120572-Proteobacteria Caulobacteraceae 1 (303)


120574-Proteobacteria Enterobacteriaceae 10 (3030)Flavobacteriia Myroidaceae 1 (303)

Uncultured bacteria clone 2 (606)


Total treated water 33

Municipal taps



120574-Proteobacteria Enterobacteriaceae 21 (3088)Moraxellaceae 6 (882)

Flavobacteriia Myroidaceae 3 (441)Uncultured bacteria clone


Total municipal tap 68Note identification was based on 16S rDNA sequencing These bacteria were obtained from our previous works [3ndash5]

raw final and municipal tap sources The results showedthat even among bacteria from the municipal tap and finaltreated water from the dam which are the point of consumerconsumption 119887119897119886TEM and 119887119897119886CTX occurred among bacteriafrom these sources in high number Xi et al [13] also observedselective increases in the levels of both genes in tap waterdue to either water treatment or regrowth within drinkingwater distribution systems This as they therefore reportedsuggested the spread of at least some beta-lactam-resistantdeterminants through drinking water distribution systemsHowever in this study it is important to point out that everysite is different for numerous variables making it impossibleto derive meaningful correlations between water treatmentpractices and the occurrence of beta-lactamase resistancegenes Given these differences the only practical meansto assess the effects of water treatment practices (whichis not our goal with this paper) would be to test changeswith experimental manipulation We have been reluctant to

provide significant detail of the sample sites precisely becausewe do not wish to imply that there are defendable correlationsbetween occurrence and site characteristics nor do we wishto encourage readers to draw such inferences

Moreover we observed that there were beta-lactam resis-tant strains that were negative for the PCR assays used in thisstudy The most commonly detected bla genes were 119887119897119886TEMand 119887119897119886SHV among Klebsiella This finding is contrary toprevious reports [14ndash16]They observed dominance of 119887119897119886CTXamong non-TEM and SHV bacteria from clinical environ-ment which were similar to what Ojdana et al [17] reportedamong clinical samples from Poland Additionally studieson Pseudomonas spp isolated from these water distributionsystems also observed a higher occurrence of 119887119897119886TEM (409)and 119887119897119886CTX (273) while none of the pseudomonads showedthe presence of 119887119897119886CTX [18] Our observation of the highestoccurrence of bla gene among Gram-negative bacteria whencompared to Gram-positive bacteria in this study is similar


Table 2 Characterization of a number of cultured bacterial isolates encoding different ESBL genotypes

Genusspeciesaccession number Source Resistant phenotypes blaTEM blaSHV blaCTXDam 1a

Escherichia coli AP0109601 DAM 1IRW T AM S C N SXT SU blaTEM

Uncultured bacterium clone JN5957831 DAM 1IFFW T FF AM G SU blaTEM

Bacillus thuringiensis JN3777821 DAM 1IFFW

SU AM T E SXT RIF LINGEN blaTEM

Brevundimonas diminuta EU5453971 DAM 1IFFW S G K N AM SXT SU blaSHV

Proteus mirabilis AB6261231 DAM 1IFFW

FF T S G K C AMC AM SUSXT blaTEM

Bacillus thuringiensis JN3777821 DAM 1IFM1 SU AM E SXT RIF LIN blaTEM blaSHV

Dam 2b

Bacillus altitudinisHQ4328111 DAM 2EDRW SU E RIF LIN AM blaSHV

Bordetella sp HQ8407201 DAM 2EDRW T FF S C N CEF AM SXT SU blaTEM

Proteus vulgaris JN6308881 DAM 2EDRW T AM SXT SU blaTEM

Staphylococcus sp JN6957101 DAM 2EDRW SU T E SXT RIF LIN AM blaTEM

Stenotrophomonas maltophilia JN7037321 DAM 2EDRW T S K CEF AM AMC SU blaTEM blaSHV

Bacillus cereus AP0072091 DAM 2EDFW SU AM T E SXT RIF LIN blaTEM

Morganella sp GQ1797061 DAM 2EDFW T S AM SXT SU blaTEM

Psychrobacter spHQ7306971 DAM 2EDM2 T S CEF AM SXT SU blaTEM

Dam 3c

Alcaligenes faecalis JN1621241 DAM 3ARW S CEF AM SXT SU blaSHV

Klebsiella pneumoniae AB6756001 DAM 3ARW

FF T S C AMC CEF AM SUSXT blaTEM

Leucobacter komagatae AJ7463371 DAM 3ARW T S AM G K SXT N SU blaTEM

Proteus mirabilis AB6261231 DAM 3ARW T S AM N SXT SU blaTEM

Uncultured bacterium clone JN5957831 DAM 3ARW

T G K C N CEF AM SXTAMC SU blaTEM

Bacillus pumilus EF0106731 DAM 3AFW SU AM T E SXT RIF LIN blaTEM

Klebsiella pneumoniae JF9199091 DAM 3AFW T S C AM SXT SU blaSHV

Myroides odoratus AB5177091 DAM 3AFW

FF T S G K C AM SXT AMCSU blaTEM

Proteus vulgaris JN6308881 DAM 3AFW

FF T S C N CEF AM SXTAMC SU blaTEM

Acinetobacter calcoaceticus DAM 3AM2 S AMC AM SU blaTEM blaSHV

Chromobacterium sp AB4261181 DAM 3AM1

T S CEF AM SXT SU AMCSU blaTEM

Klebsiella pneumoniae JF5131711 DAM 3AM2

FF C CEF AM SXT AMC SUAMC SU blaTEM blaSHV


Table 2 Continued

Genusspeciesaccession number Source Resistant phenotypes blaTEM blaSHV blaCTXDam 4d

Aeromonas caviae AB6261321 DAM 4ERW T S AM SXT N AMC SU blaTEM

Alcaligenes faecalisHQ1617771 DAM 4ERW T S K AM SU blaTEM

Alcaligenes faecalis JN1621241 DAM 4ERW T S K AM SU blaTEM

Klebsiella pneumoniae JN5450391 DAM 4ERW S CEF AM SXT AMC SU blaTEM

Klebsiella pneumoniae JN5450391 DAM 4ERW T K N AM SU blaTEM blaSHV

Klebsiella pneumoniae JF5131721 DAM 4ERW T FF S C AM SXT AMC SU blaTEM blaSHV

Morganella morganii FJ9718681 DAM 4ERW T S K CEF AM SXT AMC SU blaTEM

Proteus vulgaris JN6308881 DAM 4ERW T C CEF AM blaTEM

Proteus mirabilis GU4209881 DAM 4ERW T S K N AM SXT AMC SU blaTEM

Proteus vulgaris JN6308881 DAM 4ERW

FF T S G K C AM SXT NAMC SU blaTEM

Providencia vermicola NR 0424151 DAM 4ERW T G AM SU blaTEM

Trabulsiella guamensis AB2737371 DAM 4ERW T C CEF AM SU SXT blaSHV

Dam 5e

Klebsiella sp JN0364331 DAM 5OWODFW T FF S C AM SXT AMC SU blaTEM blaSHV

Alcaligenes faecalis JN1621241 DAM 5OWODM2 T S G K C AM SXT SU blaTEM

Escherichia coli CP0030341 DAM 5OWODM2 T AM AMC SU blaTEM


Morganella morganii AM9312641 DAM 5OWODM1 T S CEF AM SXT AMC SU blaTEM

Morganella morganii AB0892451 DAM 5OWODM3 T S K CEF SXT AMC SU blaTEM

Myroides odoratus AB5177091 DAM 5OWODM3

T S G K CEF AM SXT AMCSU blaTEM

Serratia marcescens FJ6079821 DAM 5OWODM3 T AM AMC CEF SU blaSHV

Dam 6f

Acinetobacter baumannii JF9198661 DAM 6OWIRW T FF S C AM SXT AMC SU blaTEM

Bacillus thuringiensis JN3777821 DAM 6OWIRW

SU AM T SXT RIF LIN CIPGEN blaTEM blaSHV

Klebsiella sp DQ9892152 DAM 6OWIRW T S AM SXT SU blaTEM blaCTX

Klebsiella pneumoniae CP0029101 DAM 6OWIRW T S C AM SXT AMC SU blaTEM blaSHV


Table 2 Continued

Genusspeciesaccession number Source Resistant phenotypes blaTEM blaSHV blaCTX

Klebsiella oxytoca JF3173501 DAM 6OWIRW K AM SXT SU blaCTX

Morganella morganii AB0892451 DAM 6OWIRW T S AM AMC SU blaCTX

Proteus vulgaris JN6308881 DAM 6OWIRW

T FF S C CEF SXT N AMCSU blaTEM

Serratia marcescens JF4299361 DAM 6OWIRW T S C CEF AM AMC SU blaSHV

Uncultured bacterium JN5950681 DAM 6OWIRW S G K AM SU blaTEM

Bacillus altitudinisHQ4328111 DAM 6OWIFW SU AM T E SXT RIF LIN blaTEM blaSHV

Alcaligenes sp JF7076021 DAM 6OWIM1 T S G K CEF AM AMC SU blaTEM

Alcaligenes faecalisHM1458961 DAM 6OWIM1 T S G K CEF AM AMC SU blaTEM blaSHV

Alcaligenes sp JF3038931 DAM 6OWIM2 T S G K N CEF AM SU blaSHV

Citrobacter freundii JN6445671 DAM 6OWIM1 T S AM SXT N AMC SU blaTEM

Klebsiella pneumoniae CP0029101 DAM 6OWIM1 T S CEF AM SU blaSHV

Proteus mirabilis AB6261231 DAM 6OWIM2 T S C N AMC SXT blaSHV

Codes aObafemi Awolowo University Ife Osun State IRW = Ife raw water IFFW = Ife treated water IFM1 = Ife municipal tap 1 IFM2 = Ife municipal tap2 bEde Osun State EDRW = Ede raw water EDFW = Ede treated water EDM1 = Ede municipal tap 1 EDM2 = Ede municipal tap 2 cAsejire Oyo StateNigeria ARW = Asejire raw water AFW = Asejire treated water AM1 = Asejire municipal tap 1 AM2 = Asejire municipal tap 2 dEleyele Oyo State ERW= Eleyele raw water EFW = Eleyele treated water EM1 = Eleyele municipal tap 1 EM2 = Eleyele municipal tap 2 eOwena Ondo Ondo State OWODRW =Owena Ondo raw water OWODFW = Owena Ondo treated water OWODM1 = Owena ondo municipal tap 1 OWODM2 = Owena Ondo municipal tap 2fOwena-Idanre Ondo State OWIRW=Owena-Idanre raw water OWIFW=Owena-Idanre treated water OWIM1 =Owena-Idanre municipal tap 1 OWIM2= Owena-Idanre municipal tap 2 Note bacteria was identified to the genus level by 16S rDNA partial sequenceAmpicillin (AM) ceftiofur (CEF) chloramphenicol (C) florfenicol (FF) kanamycin (K) streptomycin (S) gentamycin (GEN) tetracycline (T) nalidixic acid(N) sulfamethoxazole (SU) sulfamethoxazoletrimethoprim (SXT) amoxicillinclavulanic acid (AMC) erythromycin (E) rifampin (RIF) lincomycin (LIN)ciprofloxacin (CIP)

to reports of [19 20] These authors also confirm 119887119897119886TEM thatwas frequently detected among Gram-negative bacteria fromthis study as the most common bla gene in their studies

In this study environmental bacteria belonging to each ofthese genera Bordetella Brevundimonas ChromobacteriumProvidencia Psychrobacter Stenotrophomonas Trabulsiellaand Aeromonas possess at least one of the beta-lactamaseresistance genes tested the most common among them is119887119897119886TEM Occurrence of this gene in these environmentalisolates is contrary to the report that ESBL production ismostly found to occur among enteric species [21] The firstbla genes (119887119897119886BOR-1 and 119887119897119886OXA-2) were reported in Bordetellaby Kadlec et al [22] However we did not come acrossany publication where 119887119897119886TEM has been reported in thisbacterium This could be the first report of this gene in thisbacterium Nevertheless 119887119897119886TEM has been reported in Prov-idencia [23] Stenotrophomonas [24] and Aeromonas [25]In fact another bla gene such as 119887119897119886LI has been reported inStenotrophomonas from China [26] and 119887119897119886SHV and 119887119897119886CTX-Mhave been reported in Aeromonas [25] Moreover no 119887119897119886SHV

has also been reported in Trabulsiella this report probablymight be its first description

The association of more than one 120573-lactamase within thesame isolate has been reported [27 28] However from ourstudies the most common of this association is 119887119897119886SHV +119887119897119886TEM This was detected among Acinetobacter AlcaligenesBacillus Klebsiella and Stenotrophomonas while the combi-nation of 119887119897119886CTX and 119887119897119886TEM was only observed in KlebsiellaThis occurrence denotes the wider dissemination of thesebla genes probably due to involvement of genetic element inmobilization of these genes [29]These same authors [29] alsoobserved various combinations of 119887119897119886CTX 119887119897119886TEM and 119887119897119886SHVin Klebsiella from clinical isolates in India

The occurrence of ESBL genes among bacteria from thisstudy has a public health implication Previous studies haveshown that potential ESBL species such as K pneumoniathe most frequent bacterium with bla gene in this studyand E coli have a high tendency to possess and transfer blagenes [30] However this may occur through conjugationbecause the genes are often found in mobile elements like


Table 3 Number of bacteria and genes observed from all raw treated and municipal taps carrying at least one bla gene tested for in thisstudy

Genus bla genes from raw water bla genes from final water bla genes from municipal tapsBacterianumber blaTEM blaSHV blaCTX

Bacterianumber blaTEM blaSHV blaCTX


Acinetobacter spp 1 1 0 0 0 0 0 0 1 1 1 0Aeromonas spp 1 1 0 0 0 0 0 0 0 0 0 0Alcaligenes spp 3 2 1 0 0 0 0 0 4 3 1 0Bacillus spp 2 1 2 0 4 4 1 0 1 1 1 0Bordetella spp 1 1 0 0 0 0 0 0 0 0 0 0Brevundimonas spp 0 0 0 0 1 0 1 0 0 0 0 0Chromobacterium spp 0 0 0 0 0 0 0 0 1 1 0 0Citrobacter spp 0 0 0 0 0 0 0 0 1 1 0 0E coli 1 1 0 0 0 0 0 0 2 2 0 0Klebsiella spp 7 6 3 1 2 0 1 0 2 1 2 0Leucobacter spp 1 1 0 0 0 0 0 0 0 0 0 0Morganella spp 2 1 0 1 1 1 0 0 2 2 0 0Myroides spp 0 0 0 0 1 1 0 0 1 1 0 0Proteus spp 6 6 0 0 2 2 0 0 1 0 1 0Providencia spp 1 1 0 0 0 0 0 0 0 0 0 0Psychrobacter spp 0 0 0 0 0 0 0 0 1 1 0 0Serratia spp 1 0 1 0 0 0 0 0 1 0 1 0Staphylococcus spp 1 1 0 0 0 0 0 0 0 0 0 0Stenotrophomonas spp 1 1 1 0 0 0 0 0 0 0 0 0Trabulsiella spp 1 0 1 0 0 0 0 0 0 0 0 0Uncultured bacteria clone 2 2 0 0 1 1 0 0 0 0 0 0Total 32 26 9 2 12 9 3 0 18 14 7 0

transposons and integrons [31] Some of these species maybe pathogenic strains that have the potential to cause life-threatening diseases and widespread outbreak For instanceZhang et al [32] have reported that 119887119897119886CTX-M and 119887119897119886TEMgenes in opportunistically pathogenic Klebsiella spp havebeen associated with nosocomial infections and outbreak ofdiarrheaTherefore occurrence of these bacteria especially inthe drinking water poses a lot of danger to health economyand social well-being of consumersThis populace could alsobe exposed to these genes carrying pathogenic species infood and food products by the use of the contaminated waterfor domestic purposes farming and agriculture It shouldalso be noted that the fact that these species are multidrugresistance deepens the gravity of the situation However fromour observation during sampling the possible source of theseMDR bacteria especially in the raw water could be from run-off from agricultural farmlands located very close to some ofthese constructed dams [4] Some of these farmlands makeuse of organic fertilizers whichmay consist of unmetabolizedantibiotics which may eventually get to the water throughrun-off which may cause selective pressure on the bacteriain the aquatic systems

From the bacteria found in Nigeria many studies havedescribed the occurrence of bla genes among clinical isolatesFor example Akujobi et al [33] reported 119887119897119886TEM in E coli

while Akinniyi et al [34] reported the gene not only in Ecoli but also among Klebsiella Salmonella Citrobacter Enter-obacter Pseudomonas and Proteus The highest prevalence(56) was also in Klebsiella which is similar to our findingsHowever from environmental isolates few studies seem tohave been conducted Moreover Adelowo et al [35] reported119887119897119886TEM in E coli from well water while Chikwendu et al [36]described not only 119887119897119886TEM among Pseudomonas from riverand aquaculture samples but also 119887119897119886SHV Moreover thisstudy seems to be the first report describing these genesamong a wide diversity of environmental bacteria fromNigeria drinking water distribution systems It is thereforeimportant to raise public and health worker awarenessin terms of prevention of outbreak of MDR infectiouspathogens among consumersThis also undermines the needfor government agencies controlling these dams and healthorganizations to initiate measures to effectively control therelease of contaminant into the environment It would also begood to continually isolate bacteria from other water distri-bution systems in Nigeria and to carry out further moleculartesting for the presence of other bla genes characterize anddetermine whether these genes are present on transferableplasmids transposons or integrons which would enhanceeasy spreading


5 Conclusion

The occurrence of beta-lactamase producing bacteria andgenes in all sampled water in this study especially treateddrinking water showed that these water distribution systemscould serve as a vehicle for transmission of these antibioticresistance bacteria and genes to consumers hence of a greatpublic health concern

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Ayodele T Adesoji and Adeniyi A Ogunjobi planned thisstudy Ayodele T Adesoji performed the experiment underthe guidance of Adeniyi A Ogunjobi Ayodele T Adesojiwrote the paper All authors read and approved the finalpaper

Acknowledgments

The authors acknowledge Dr Call Douglas of Paul GAllen School of Global Animal Health Washington StateUniversity USA who allowed themolecular characterizationof the bacteria isolates to be carried out in his laboratory andLisa Lorfe who supplied the technical knowhow

References

[1] N J Ashbolt ldquoMicrobial contamination of drinking water anddisease outcomes in developing regionsrdquo Toxicology vol 198no 1ndash3 pp 229ndash238 2004

[2] 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

[3] A T Adesoji and A A Ogunjobi ldquoOccurrence of multidrug-resistant bacteria in selected water distribution systems in OyoState Nigeriardquo Global Veterinaria vol 11 no 2 pp 214ndash2242013

[4] A A Timi and O A Adeniyi ldquoPhysicochemical properties andoccurrence of antibiotic-resistant bacteria in Ife and Ede waterdistribution systems of southwestern Nigeriardquo World AppliedSciences Journal vol 27 no 9 pp 1098ndash1110 2013

[5] A T Adesoji A A Ogunjobi and I O Olatoye ldquoDrinkingwater distribution systems of dams in Ondo State Nigeriaas reservoir of multi-drug resistant bacteriardquo World AppliedSciences Journal vol 32 pp 403ndash414 2014

[6] K Bush ldquoCharacterization of 120573-lactamasesrdquo AntimicrobialAgents and Chemotherapy vol 33 no 3 pp 259ndash263 1989

[7] G S Singh ldquo120573-lactam in the newmillennium Part-Imonobac-tams and carbapenemsrdquoMini-Reviews in Medicinal Chemistryvol 4 no 1 pp 69ndash92 2004

[8] K Kumar S C Gupta Y Chander and A K Singh ldquoAntibioticuse in agriculture and its impact on the terrestrial environmentrdquoAdvances in Agronomy vol 87 pp 1ndash54 2005

[9] P A Bradford ldquoExtended-spectrum 120573-lactamases in the 21stcentury characterization epidemiology and detection of this

important resistance threatrdquo Clinical Microbiology Reviews vol14 no 4 pp 933ndash951 2001

[10] L Brusetti T Glad S Borin et al ldquoLow prevalence of 119887119897119886TEMgenes in Arctic environments and agricultural soil and rhizo-sphererdquo Microbial Ecology in Health and Disease vol 20 no 1pp 27ndash36 2008

[11] A T Adesoji A A Ogunjobi I O Olatoye and D R DouglasldquoPrevalence of tetracycline resistance genes among multi-drugresistant bacteria from selected water distribution systems insouthwestern Nigeriardquo Annals of Clinical Microbiology andAntimicrobials vol 14 article 35 2015

[12] I S Henriques F Fonseca A Alves M J Saavedra andA Correia ldquoOccurrence and diversity of integrons and 120573-lactamase genes among ampicillin-resistant isolates from estu-arine watersrdquo Research in Microbiology vol 157 no 10 pp 938ndash947 2006

[13] C Xi Y Zhang C F Marrs et al ldquoPrevalence of antibioticresistance in drinking water treatment and distribution sys-temsrdquo Applied and Environmental Microbiology vol 75 no 17pp 5714ndash5718 2009

[14] U Govinden C Mocktar P Moodley A W Sturm and S YEssack ldquoGeographical evolution of the CTX-M 120573-lactamase anupdaterdquo African Journal of Biotechnology vol 6 no 7 pp 831ndash839 2007

[15] C Dallenne A da Costa D Decre C Favier and G ArletldquoDevelopment of a set of multiplex PCR assays for the detectionof genes encoding important 120573-lactamases in Enterobacteri-aceaerdquo Journal of Antimicrobial Chemotherapy vol 65 no 3 pp490ndash495 2010

[16] S-Y Lu Y-L Zhang S-N Geng et al ldquoHigh diversity ofextended-spectrum beta-lactamase-producing bacteria in anurban river sediment habitatrdquo Applied and EnvironmentalMicrobiology vol 76 no 17 pp 5972ndash5976 2010

[17] D Ojdana P Sacha P Wieczorek et al ldquoThe occurrence of119887119897119886CTX-M 119887119897119886SHV and 119887119897119886TEM genes in extended-spectrum 120573-lactamase-positive strains ofKlebsiella pneumoniae Escherichiacoli and Proteus mirabilis in Polandrdquo International Journal ofAntibiotics vol 2014 Article ID 935842 7 pages 2014

[18] A T Adesoji A A Ogunjobi and I O Olatoye ldquoMolecularcharacterization of selected multidrug resistant Pseudomonasfrom water distribution systems in southwestern NigeriardquoAnnals of Clinical Microbiology and Antimicrobials vol 14article 39 2015

[19] F Luzzaro M Mezzatesta C Mugnaioli et al ldquoTrends inproduction of extended-spectrum 120573-lactamases among enter-obacteria of medical interest report of the second Italiannationwide surveyrdquo Journal of Clinical Microbiology vol 44 no5 pp 1659ndash1664 2006

[20] T Spanu F Luzzaro M Perilli et al ldquoOccurrence of extended-spectrum 120573-lactamases inmembers of the family Enterobacteri-aceae in Italy implications for resistance to120573-lactams and otherantimicrobial drugsrdquo Antimicrobial Agents and Chemotherapyvol 46 no 1 pp 196ndash202 2002

[21] S Tissera and S M Lee ldquoIsolation of extended spectrum 120573-lactamase (ESBL) producing bacteria fromurban surfacewatersin MalaysiardquoMalaysian Journal of Medical Sciences vol 20 no3 pp 14ndash22 2013

[22] K Kadlec I Wiegand C Kehrenberg and S Schwarz ldquoStudieson the mechanisms of 120573-lactam resistance in Bordetella bron-chisepticardquo Journal of Antimicrobial Chemotherapy vol 59 no3 pp 396ndash402 2007


[23] S Mahrouki H Chihi A Bourouis et al ldquoNosocomial dissem-ination of plasmids carrying 119887119897119886TEM-24 119887119897119886DHA-1 aac(61015840)-Ib-crand qnrA6 in Providencia spp strains isolated from a TunisianhospitalrdquoDiagnosticMicrobiology and Infectious Disease vol 81no 1 pp 50ndash52 2015

[24] M B Avison C J von Heldreich C S Higgins P M Bennettand T R Walsh ldquoA TEM-2 120573-lactamase encoded on anactive Tn1-like transposon in the genome of a clinical isolateof Stenotrophomonas maltophiliardquo Journal of AntimicrobialChemotherapy vol 46 no 6 pp 879ndash884 2000

[25] L C Balsalobre M Dropa D E de Oliveira et al ldquoPresenceof 119887119897119886TEM-116 gene in environmental isolates of Aeromonashydrophila and Aeromonas jandaei from Brazilrdquo Brazilian Jour-nal of Microbiology vol 41 no 3 pp 718ndash719 2010

[26] Z Yang W Liu Q Cui et al ldquoPrevalence and detectionof Stenotrophomonas maltophilia carrying metallo-120573-lactamaseblaL1 in Beijing Chinardquo Frontiers in Microbiology vol 5 article692 2014

[27] A Karim L Poirel S Nagarajan and P Nordmann ldquoPlasmid-mediated extended-spectrum 120573-lactamase (CTX-M-3 like)from India and gene association with insertion sequenceISEcp1rdquo FEMSMicrobiology Letters vol 201 no 2 pp 237ndash2412001

[28] C J Munday G M Whitehead N J Todd M Camp-bell and P M Hawkey ldquoPredominance and genetic diver-sity of community- and hospital-acquired CTX-M extended-spectrum 120573-lactamases in York UKrdquo Journal of AntimicrobialChemotherapy vol 54 no 3 pp 628ndash633 2004

[29] M ShahidA Singh F Sobia et al ldquo119887119897119886CTX-M 119887119897119886TEM and 119887119897119886SHVin Enterobacteriaceae fromNorth-Indian tertiary hospital highoccurrence of combination genesrdquo Asian Pacific Journal ofTropical Medicine vol 4 no 2 pp 101ndash105 2011

[30] J K Bailey J L Pinyon S Anantham and R M HallldquoDistribution of the 119887119897119886TEM gene and 119887119897119886TEM-containing trans-posons in commensal Escherichia colirdquo Journal of AntimicrobialChemotherapy vol 66 no 4 pp 745ndash751 2011

[31] M LGuerri AAladuena A Echeıta andR Rotger ldquoDetectionof integrons and antibiotic-resistance genes in Salmonella enter-ica serovar Typhimurium isolates with resistance to ampicillinand variable susceptibility to amoxicillin-clavulanaterdquo Interna-tional Journal of Antimicrobial Agents vol 24 no 4 pp 327ndash3332004

[32] Y Zhang H Zhou X-Q Shen P Shen Y-S Yu and L-J LildquoPlasmid-borne armAmethylase gene togetherwith 119887119897119886CTX-M-15and 119887119897119886TEM-1 in a Klebsiella oxytoca isolate from Chinardquo Journalof Medical Microbiology vol 57 no 10 pp 1273ndash1276 2008

[33] C N Akujobi C C Ezeanya and N I Aghanya ldquoDetectionof cefotaximase genes of beta lactamase among clinical isolatesof Escherichia coli in a university teaching hospital NigeriardquoJournal of Medical Sciences vol 12 no 7 pp 244ndash247 2012

[34] A P Akinniyi O Afolabi B A Iwalokun E Oluwaseun andK O Onagbesan ldquoClonal dissemination of 119887119897119886TEM 120573-lactamasestrains among enteric isolates in Abeokuta Nigeriardquo ResearchJournal of Microbiology vol 6 no 12 pp 919ndash925 2011

[35] O O Adelowo O E Fagade and Y Agersoslash ldquoAntibioticresistance and resistance genes in Escherichia coli from poultryfarms southwest Nigeriardquo Journal of Infection in DevelopingCountries vol 8 no 9 pp 1103ndash1112 2014

[36] C I Chikwendu S N Ibe and G C Okpokwasili ldquoDetectionof 119887119897119886SHV and 119887119897119886TEM beta-lactamase genes in multi-resistantPseudomonas isolates from environmental sourcesrdquo AfricanJournal of Microbiology Research vol 5 no 15 pp 2067ndash20742011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


substrate range (httpwwwlaheyorgStudiestemtableasp)[10]

Previous reports indicated that multidrug resistant bac-teria are present in drinking water distribution systemsfrom southwestern Nigeria [3ndash5] These bacteria encodedresistance to a diversity of beta-lactams including ceftiofurampicillin and combination of amoxicillin and amoxicillinclavulanic acid

The aim of this study was to genotype MDR bacteriaisolated from our previous studies [3ndash5] for the presence ofselected beta-lactamase resistance genes using PCR

2 Materials and Methods

21 Dam Description Sampling Selection Isolation Storageand Molecular Characterization of Bacteria The descriptionof sampled dams in this study is in our previous publications[3ndash5] Moreover for clarity of this paper ninety-six watersamples were purposively collected aseptically into sterilescrew cap bottles from six selected water distribution sys-tems of dams in Ife Ede Asejire Eleyele Owena Ondoand Owena-Idanre in southwestern Nigeria Samples werecollected four times between December 2010 and July 2011from raw treated and two randomly selected municipaldistribution taps Afterwards samples were serially dilutedand plated on Nutrient agar eosin methylene blue agar(EMB) and Deoxycholate agar (DCA) Thereafter bacteriawere picked with the aim of maximizing the diversity ofcolony morphology represented from each sample Pickedcolonies were restreaked on Nutrient agar to obtain purecultures These were subsequently transferred to Nutrientagar slants and also stored in phosphate buffer glycerol atminus80∘C [3ndash5]Molecular characterization of bacteria using 16SrDNA sequencing was determined as described in Adesoji etal [11]

22 Antibiotic Susceptibility Testing Agar dilution assays(also called breakpoint assays) were conducted using Luria-Bertani agar with seeded antibiotics used to assess antibioticsusceptibility Antibiotics concentrations used for Gram-negative bacteria included florfenicol (16120583gmL) tetracy-cline (16 120583gmL) streptomycin (16 120583gmL) gentamycin(16 120583gmL) kanamycin (64 120583gmL) chloramphenicol(32 120583gmL) nalidixic acid (30 120583gmL) amoxicillinclavulanicacid (3216 120583gmL) ceftiofur (12120583gmL) sulfamethoxazole(512120583gmL) and sulfamethoxazoletrimethoprim (764 120583gmL) Antibiotics concentrations used for Gram-positivebacteria include sulfamethoxazole (512120583gmL) ampicillin(05 120583gmL) tetracycline (16 120583gmL) sulfamethoxazoletri-methoprim (764120583gmL) gentamycin (16 120583gmL) erythro-mycin (8120583gmL) rifampin (4120583gmL) lincomycin (4120583gmL)and ciprofloxacin (4 120583gmL) Negative and positive controlsused were E coli strain K12 and E coli strain H4H respec-tively as we described in our previous studies [3ndash5 11]

23 Resistance Genotyping PCR testing was conducted forbacteria having resistance to ge3 classes of antibiotics includ-ing resistance to amoxicillinclavulanic acid ceftiofur orampicillinThereafter forward and reverse primer specific for

selected ESBL genes included 119887119897119886SHV (SHV F 51015840-GCGAAA-GCCAGCTGTCGGGC-31015840 and SHV R 51015840-GATTGGCGG-CGCTGTTATCGC-3) 119887119897119886CTX M (CTX F 51015840-GTGCAG-TACCAGTAAAGTTATGG-31015840 and CTX R 51015840-CGCAAT-ATCATTGGTGGTGCC-31015840) and 119887119897119886TEM (TEM F 51015840-AAA-GATGCTGAAGATCA-31015840 and TEM R 51015840-TTTGGTATG-GCTTCATTC-31015840) [12] Condition for 119887119897119886SHV PCR included1min denaturation (95∘C followed by 30 cycles of 96∘C for30 s 62∘C for 30 s and 72∘C for 30 s and final extension of72∘C for 10min Conditions were identical for other assaysexcept the annealing temperatures which were 55∘C and44∘C for 119887119897119886CTX-M and 119887119897119886TEM respectively Afterwards PCRproducts were separated sized and visualized by using 1agarose gel electrophoresis to confirm amplification

3 Results

31 Bacteria Isolates Isolates used in this study wereselected from our previous studies [3ndash5] and represented120572-Proteobacteria 120573-Proteobacteria 120574-ProteobacteriaFlavobacteriia Bacilli and Actinobacteria with 33 66 and68 being isolated from all treated raw and municipal tapsrespectively (Table 1) Proteus was the most frequent (1818)isolated Gram-negative genus from the treated water whileKlebsiella was the most frequently (1515) isolated genusfrom raw water Bacillus was the most common isolatedGram-positive genus for treated and municipal water

32 PCR-Positive Isolates In this study 61 isolates out of164 MDR isolates were PCR-positive for at least one tar-geted gene Highest occurrence of bla gene among Gram-negative bacteria compared to Gram-positive bacteria wasobserved Most commonly isolated genus carrying bla geneis Klebsiella (1803) followed by Proteus spp (1475)119861119897119886TEM was detected in the majority of beta-lactam resis-tant isolates (5061) while 119887119897119886CTX was rarely detected (361)(Table 2) A combination of two genes 119887119897119886SHV + 119887119897119886TEMor 119887119897119886CTX + 119887119897119886TEM was observed in 11 and 1 bacteriarespectively (Table 2) Other genera including AquitaleaComamonas Enterobacter Leucobacter Lysinibacillus Pan-toea Pseudochrobactrum Sphingobacterium and Ralstoniawere tested but were PCR-negative for resistance genes

4 Discussion

The hazard associated with the pathogenicity of microbes isaggravated by its ability to resist destruction by antibiotics [2]In this study beta-lactamase producing bacteria and genes(ie 119887119897119886CTX and 119887119897119886TEM) were detected from every sampledwater distribution system This is similar to the report of Xiet al [13] who also investigated the prevalence and dynamicsof heterotrophic antibiotics resistance bacteria and genesin drinking water source and treated drinking water usingculture-dependent methods and molecular techniques Theauthors observed the presence of 119887119897119886TEM and 119887119897119886SHV genesin all water samples except one which is evidence that thesegenes are distributed widely in drinking water systems Thisis similar to what we also reported in Table 3 showing thespread of these beta-lactamase resistance genes among all




Raw water









Treated water







Municipal taps





















Dam 2b









Dam 3c




















Table 2 Continued















Dam 5e










Dam 6f







Table 2 Continued































5 Conclusion


Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Raw water









Treated water







Municipal taps





















Dam 2b









Dam 3c




















Table 2 Continued















Dam 5e










Dam 6f







Table 2 Continued































5 Conclusion


Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Dam 2b









Dam 3c




















Table 2 Continued















Dam 5e










Dam 6f







Table 2 Continued































5 Conclusion


Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 2 Continued















Dam 5e










Dam 6f







Table 2 Continued































5 Conclusion


Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 2 Continued































5 Conclusion


Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











5 Conclusion


Competing Interests




Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article detection of extended spectrum beta

Documents