# Detection of Extended Spectrum Beta-Lactamases Resistance Genes among Bacteria Isolated from Selected Drinking Water Distribution Channels in Southwestern Nigeria

Authors: Ayodele T. Adesoji; Adeniyi A. Ogunjobi
Journal: BioMed Research International
 (2016)

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## Abstract

Extended Spectrum Beta-Lactamases (ESBL) provide high level resistance to beta-lactam antibiotics among bacteria. In this study, previously described multidrug resistant bacteria from raw, treated, and municipal taps of DWDS from selected dams in southwestern Nigeria were assessed for the presence of ESBL resistance genes which includeblaTEM, blaSHV, and blaCTX by PCR amplification. A total of 164 bacteria spread across treated (33), raw (66), and municipal taps (68), belonging to α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Flavobacteriia, Bacilli, and Actinobacteria group, were selected for this study. Among these bacteria, the most commonly observed resistance was for ampicillin and amoxicillin/clavulanic acid (61 isolates). Sixty-one isolates carried at least one of the targeted ESBL genes with blaTEM being the most abundant (50/61) and blaCTX being detected least (3/61).Klebsiella was the most frequently identified genus (18.03%) to harbour ESBL gene followed byProteus (14.75%). Moreover, combinations of two ESBL genes, blaSHV+blaTEM or blaCTX+blaTEM, were observed in 11 and 1 isolate, respectively. In conclusion, classic blaTEM ESBL gene was present in multiple bacterial strains that were isolated from DWDS sources in Nigeria. These environments may serve as foci exchange of genetic traits in a diversity of Gram-negative bacteria.


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## Body

## 1. Introduction

Access to safe drinking water is essential for human health [1]. While access to safe and affordable water should be available to everyone, this remains a challenge in low- and middle-income countries including Nigeria, which is the most populous country in Africa. Safe drinking water is mostly viewed in terms of organic and inorganic contaminants, but also in terms of biological contamination. In this respect, less attention has been given to the role that water may play in the dissemination of antibiotic resistance traits in populations that are exposed to substandard water on a daily basis [2–5].Arguably, the most clinically important antibiotic resistance genes are those that encode enzymes that hydrolyzeβ-lactams (bla genes) [6]. These traits confer high level resistance to β-lactam antibiotics, which are the most widely used antibiotics in clinical and veterinary practice [7, 8]. Extended Spectrum β-Lactamase (ESBL) is group of enzymes that can hydrolyze a variety of β-lactams including cephalosporins like ceftazidime, cefotaxime, and ceftriaxone and monobactams like aztreonam in addition to penicillin but does not hydrolyze cephamycins like cefoxitin. Most of the ESBL also have the ability to hydrolyze fourth-generation cephalosporins including cefepime [9].A variety of transferable genes encodingβ-lactamase activity have been described in clinical environments including blaCTX-M, blaGES, blaHER, blaOXA, blaOXY, blaSED, blaSHV, blaSPM, blaVEB, blaVIM, andampC[10]. Among the most commonbla genes is the blaTEM-1 gene, the first describedbla gene and a representative of the blaTEM group that now consists of more than 220 different distinct variants (“alleles”), which encode different amino acid polymorphisms that extend their substrate range (http://www.lahey.org/Studies/temtable.asp) [10].Previous reports indicated that multidrug resistant bacteria are present in drinking water distribution systems from southwestern Nigeria [3–5]. These bacteria encoded resistance to a diversity of beta-lactams including ceftiofur, ampicillin, and combination of amoxicillin and amoxicillin/clavulanic acid.The aim of this study was to genotype MDR bacteria isolated from our previous studies [3–5] for the presence of selected beta-lactamase resistance genes using PCR.


## 2. Materials and Methods

### 2.1. Dam Description, Sampling, Selection, Isolation, Storage, and Molecular Characterization of Bacteria

The description of sampled dams in this study is in our previous publications [3–5]. Moreover, for clarity of this paper, ninety-six water samples were purposively collected aseptically into sterile screw cap bottles from six selected water distribution systems of dams in Ife, Ede, Asejire, Eleyele, Owena Ondo, and Owena-Idanre in southwestern Nigeria. Samples were collected four times between December 2010 and July 2011 from raw, treated, and two randomly selected municipal distribution taps. Afterwards, samples were serially diluted and plated on Nutrient agar, eosin methylene blue agar (EMB), and Deoxycholate agar (DCA). Thereafter, bacteria were picked with the aim of maximizing the diversity of colony morphology represented from each sample. Picked colonies were restreaked on Nutrient agar to obtain pure cultures. These were subsequently transferred to Nutrient agar slants and also stored in phosphate buffer glycerol at −80°C [3–5]. Molecular characterization of bacteria using 16S rDNA sequencing was determined as described in Adesoji et al. [11].


### 2.2. Antibiotic Susceptibility Testing

Agar dilution assays (also called breakpoint assays) were conducted using Luria-Bertani agar with seeded antibiotics used to assess antibiotic susceptibility. Antibiotics concentrations used for Gram-negative bacteria included florfenicol (16μg/mL), tetracycline (16 μg/mL), streptomycin (16 μg/mL), gentamycin (16 μg/mL), kanamycin (64 μg/mL), chloramphenicol (32 μg/mL), nalidixic acid (30 μg/mL), amoxicillin/clavulanic acid (32/16 μg/mL), ceftiofur (12 μg/mL), sulfamethoxazole (512 μg/mL), and sulfamethoxazole/trimethoprim (76/4 μg/mL). Antibiotics concentrations used for Gram-positive bacteria include sulfamethoxazole (512 μg/mL), ampicillin (0.5 μg/mL), tetracycline (16 μg/mL), sulfamethoxazole/trimethoprim (76/4 μg/mL), gentamycin (16 μg/mL), erythromycin (8 μg/mL), rifampin (4 μg/mL), lincomycin (4 μg/mL), and ciprofloxacin (4 μg/mL). Negative and positive controls used wereE. coli strain K12 andE. coli strain H4H, respectively, as we described in our previous studies [3–5, 11].


### 2.3. Resistance Genotyping

PCR testing was conducted for bacteria having resistance to ≥3 classes of antibiotics including resistance to amoxicillin/clavulanic acid, ceftiofur, or ampicillin. Thereafter, forward and reverse primer specific for selected ESBL genes includedblaSHV (SHV_F, 5′-GCGAAAGCCAGCTGTCGGGC-3′ and SHV_R, 5′-GATTGGCGGCGCTGTTATCGC-3), blaCTX_M (CTX_F, 5′-GTGCAGTACCAGTAAAGTTATGG-3′ and CTX_R, 5′-CGCAATATCATTGGTGGTGCC-3′), and blaTEM (TEM_F, 5′-AAAGATGCTGAAGATCA-3′ and TEM_R, 5′-TTTGGTATGGCTTCATTC-3′) [12]. Condition for blaSHV PCR included 1 min denaturation (95°C followed by 30 cycles of 96°C for 30 s, 62°C for 30 s, and 72°C for 30 s and final extension of 72°C for 10 min. Conditions were identical for other assays except the annealing temperatures which were 55°C and 44°C for blaCTX-M and blaTEM, respectively. Afterwards, PCR products were separated, sized, and visualized by using 1% agarose gel electrophoresis to confirm amplification.


## 2.1. Dam Description, Sampling, Selection, Isolation, Storage, and Molecular Characterization of Bacteria

The description of sampled dams in this study is in our previous publications [3–5]. Moreover, for clarity of this paper, ninety-six water samples were purposively collected aseptically into sterile screw cap bottles from six selected water distribution systems of dams in Ife, Ede, Asejire, Eleyele, Owena Ondo, and Owena-Idanre in southwestern Nigeria. Samples were collected four times between December 2010 and July 2011 from raw, treated, and two randomly selected municipal distribution taps. Afterwards, samples were serially diluted and plated on Nutrient agar, eosin methylene blue agar (EMB), and Deoxycholate agar (DCA). Thereafter, bacteria were picked with the aim of maximizing the diversity of colony morphology represented from each sample. Picked colonies were restreaked on Nutrient agar to obtain pure cultures. These were subsequently transferred to Nutrient agar slants and also stored in phosphate buffer glycerol at −80°C [3–5]. Molecular characterization of bacteria using 16S rDNA sequencing was determined as described in Adesoji et al. [11].


## 2.2. Antibiotic Susceptibility Testing

Agar dilution assays (also called breakpoint assays) were conducted using Luria-Bertani agar with seeded antibiotics used to assess antibiotic susceptibility. Antibiotics concentrations used for Gram-negative bacteria included florfenicol (16μg/mL), tetracycline (16 μg/mL), streptomycin (16 μg/mL), gentamycin (16 μg/mL), kanamycin (64 μg/mL), chloramphenicol (32 μg/mL), nalidixic acid (30 μg/mL), amoxicillin/clavulanic acid (32/16 μg/mL), ceftiofur (12 μg/mL), sulfamethoxazole (512 μg/mL), and sulfamethoxazole/trimethoprim (76/4 μg/mL). Antibiotics concentrations used for Gram-positive bacteria include sulfamethoxazole (512 μg/mL), ampicillin (0.5 μg/mL), tetracycline (16 μg/mL), sulfamethoxazole/trimethoprim (76/4 μg/mL), gentamycin (16 μg/mL), erythromycin (8 μg/mL), rifampin (4 μg/mL), lincomycin (4 μg/mL), and ciprofloxacin (4 μg/mL). Negative and positive controls used wereE. coli strain K12 andE. coli strain H4H, respectively, as we described in our previous studies [3–5, 11].


## 2.3. Resistance Genotyping

PCR testing was conducted for bacteria having resistance to ≥3 classes of antibiotics including resistance to amoxicillin/clavulanic acid, ceftiofur, or ampicillin. Thereafter, forward and reverse primer specific for selected ESBL genes includedblaSHV (SHV_F, 5′-GCGAAAGCCAGCTGTCGGGC-3′ and SHV_R, 5′-GATTGGCGGCGCTGTTATCGC-3), blaCTX_M (CTX_F, 5′-GTGCAGTACCAGTAAAGTTATGG-3′ and CTX_R, 5′-CGCAATATCATTGGTGGTGCC-3′), and blaTEM (TEM_F, 5′-AAAGATGCTGAAGATCA-3′ and TEM_R, 5′-TTTGGTATGGCTTCATTC-3′) [12]. Condition for blaSHV PCR included 1 min denaturation (95°C followed by 30 cycles of 96°C for 30 s, 62°C for 30 s, and 72°C for 30 s and final extension of 72°C for 10 min. Conditions were identical for other assays except the annealing temperatures which were 55°C and 44°C for blaCTX-M and blaTEM, respectively. Afterwards, PCR products were separated, sized, and visualized by using 1% agarose gel electrophoresis to confirm amplification.


## 3. Results

### 3.1. Bacteria Isolates

Isolates used in this study were selected from our previous studies [3–5] and represented α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Flavobacteriia, Bacilli, and Actinobacteria with 33, 66, and 68 being isolated from all treated, raw, and municipal taps, respectively (Table 1).Proteus was the most frequent (18.18%) isolated Gram-negative genus from the treated water whileKlebsiella was the most frequently (15.15%) isolated genus from raw water.Bacillus was the most common isolated Gram-positive genus for treated and municipal water.Table 1
Classes and families of selected bacteria.
Source Class Family Number (% of total from source) Raw water α-Proteobacteria Brucellaceae 1 (1.51) β-Proteobacteria Alcaligenaceae 9 (13.64) Neisseriaceae 1 (1.51) γ-Proteobacteria Enterobacteriaceae 27 (40.91) Moraxellaceae 2 (3.03) Aeromonadaceae 6 (9.09) Xanthomonadaceae 2 (3.03) Flavobacteriia Myroidaceae 1 (1.51) Uncultured bacteria clone  3 (4.55) Bacilli Bacillaceae 10 (15.15) Staphylococcaceae 3 (4.55) Actinobacteria Microbacteriaceae 1 (1.51) Total raw water  66  Treated water α-Proteobacteria Caulobacteraceae 1 (3.03) β-Proteobacteria Alcaligenaceae 5 (15.15) Neisseriaceae 1 (3.03) γ-Proteobacteria Enterobacteriaceae 10 (30.30) Flavobacteriia Myroidaceae 1 (3.03) Uncultured bacteria clone  2 (6.06) Bacilli Bacillaceae 12 (36.36) Staphylococcaceae 1 (3.03) Total treated water  33  Municipal taps α-Proteobacteria Caulobacteraceae 1 (1.47) β-Proteobacteria Alcaligenaceae 7 (10.29) Neisseriaceae 3 (4.41) γ-Proteobacteria Enterobacteriaceae 21 (30.88) Moraxellaceae 6 (8.82) Flavobacteriia Myroidaceae 3 (4.41) Uncultured bacteria clone Bacilli Bacillaceae 26 (38.24) Staphylococcaceae 1 (1.47) Total municipal tap  68 Note: identification was based on 16S rDNA sequencing. These bacteria were obtained from our previous works [3–5].


### 3.2. PCR-Positive Isolates

In this study, 61 isolates out of 164 MDR isolates were PCR-positive for at least one targeted gene. Highest occurrence ofbla gene among Gram-negative bacteria compared to Gram-positive bacteria was observed. Most commonly isolated genus carryingbla gene isKlebsiella (18.03%) followed byProteus spp. (14.75%). BlaTEM was detected in the majority of beta-lactam resistant isolates (50/61) while blaCTX was rarely detected (3/61) (Table 2). A combination of two genes, blaSHV + blaTEM or blaCTX + blaTEM, was observed in 11 and 1 bacteria, respectively (Table 2). Other genera, includingAquitalea,Comamonas,Enterobacter,Leucobacter,Lysinibacillus,Pantoea,Pseudochrobactrum,Sphingobacterium, andRalstonia, were tested but were PCR-negative for resistance genes.Table 2
Characterization of a number of cultured bacterial isolates encoding differentESBLgenotypes.
Genus/species/accession number Source Resistant phenotypes bla T E M bla S H V bla C T X Dam 1 a Escherichia coli AP010960.1 DAM 1IRW T, AM, S, C, N, SXT, SU bla T E M Uncultured bacterium clone JN595783.1 DAM 1IFFW T, FF, AM, G, SU bla T E M Bacillus thuringiensis JN377782.1 DAM 1IFFW SU, AM, T, E, SXT, RIF, LIN, GEN bla T E M Brevundimonas diminuta EU545397.1 DAM 1IFFW S, G, K, N, AM, SXT, SU  bla S H V Proteus mirabilis AB626123.1 DAM 1IFFW FF, T, S, G, K, C, AMC, AM, SU, SXT bla T E M Bacillus thuringiensis JN377782.1 DAM 1IFM1 SU, AM, E, SXT, RIF, LIN bla T E M bla S H V  Dam 2 b Bacillus altitudinis HQ432811.1 DAM 2EDRW SU, E, RIF, LIN, AM  bla S H V Bordetellasp. HQ840720.1 DAM 2EDRW T, FF, S, C, N, CEF, AM, SXT, SU bla T E M Proteus vulgaris JN630888.1 DAM 2EDRW T, AM, SXT, SU bla T E M Staphylococcussp. JN695710.1 DAM 2EDRW SU, T, E, SXT, RIF, LIN, AM bla T E M Stenotrophomonas maltophilia JN703732.1 DAM 2EDRW T, S, K, CEF, AM, AMC, SU bla T E M bla S H V Bacillus cereus AP007209.1 DAM 2EDFW SU, AM, T, E, SXT, RIF, LIN bla T E M Morganellasp. GQ179706.1 DAM 2EDFW T, S, AM, SXT, SU bla T E M Psychrobactersp. HQ730697.1 DAM 2EDM2 T, S, CEF, AM, SXT, SU bla T E M  Dam 3 c Alcaligenes faecalis JN162124.1 DAM 3ARW S, CEF, AM, SXT, SU  bla S H V Klebsiella pneumoniae AB675600.1 DAM 3ARW FF, T, S, C, AMC, CEF, AM, SU, SXT bla T E M Leucobacter komagatae AJ746337.1 DAM 3ARW T, S, AM, G, K, SXT, N, SU bla T E M Proteus mirabilis AB626123.1 DAM 3ARW T, S, AM, N, SXT, SU bla T E M Uncultured bacterium clone JN595783.1 DAM 3ARW T, G, K, C, N, CEF, AM, SXT, AMC, SU bla T E M Bacillus pumilus EF010673.1 DAM 3AFW SU, AM, T, E, SXT, RIF, LIN bla T E M Klebsiella pneumoniae JF919909.1 DAM 3AFW T, S, C, AM, SXT, SU  bla S H V Myroides odoratus AB517709.1 DAM 3AFW FF, T, S, G, K, C, AM, SXT, AMC, SU bla T E M Proteus vulgaris JN630888.1 DAM 3AFW FF, T, S, C, N, CEF, AM, SXT, AMC, SU bla T E M Acinetobacter calcoaceticus DAM 3AM2 S, AMC, AM, SU bla T E M bla S H V Chromobacteriumsp. AB426118.1 DAM 3AM1 T, S, CEF, AM, SXT, SU, AMC, SU bla T E M Klebsiella pneumoniae JF513171.1 DAM 3AM2 FF, C, CEF, AM, SXT, AMC, SU, AMC, SU bla T E M bla S H V  Dam 4 d Aeromonas caviae AB626132.1 DAM 4ERW T, S, AM, SXT, N, AMC, SU bla T E M Alcaligenes faecalis HQ161777.1 DAM 4ERW T, S, K, AM, SU bla T E M Alcaligenes faecalis JN162124.1 DAM 4ERW T, S, K, AM, SU bla T E M Klebsiella pneumoniae JN545039.1 DAM 4ERW S, CEF, AM, SXT, AMC, SU bla T E M Klebsiella pneumoniae JN545039.1 DAM 4ERW T, K, N, AM, SU bla T E M bla S H V Klebsiella pneumoniae JF513172.1 DAM 4ERW T, FF, S, C, AM, SXT, AMC, SU bla T E M bla S H V Morganella morganii FJ971868.1 DAM 4ERW T, S, K, CEF, AM, SXT, AMC, SU bla T E M Proteus vulgaris JN630888.1 DAM 4ERW T, C, CEF, AM bla T E M Proteus mirabilis GU420988.1 DAM 4ERW T, S, K, N, AM, SXT, AMC, SU bla T E M Proteus vulgaris JN630888.1 DAM 4ERW FF, T, S, G, K, C, AM, SXT, N, AMC, SU bla T E M Providencia vermicola NR_042415.1 DAM 4ERW T, G, AM, SU bla T E M Trabulsiella guamensis AB273737.1 DAM 4ERW T, C, CEF, AM, SU, SXT  bla S H V  Dam 5 e Klebsiellasp. JN036433.1 DAM 5OWODFW T, FF, S, C, AM, SXT, AMC, SU bla T E M bla S H V Alcaligenes faecalis JN162124.1 DAM 5OWODM2 T, S, G, K, C, AM, SXT, SU bla T E M Escherichia coli CP003034.1 DAM 5OWODM2 T, AM, AMC, SU bla T E M Escherichia coli CP003034.1 DAM 5OWODM2 T, AM, AMC, SU bla T E M Morganella morganii AM931264.1 DAM 5OWODM1 T, S, CEF, AM, SXT, AMC, SU bla T E M Morganella morganii AB089245.1 DAM 5OWODM3 T, S, K, CEF, SXT, AMC, SU bla T E M Myroides odoratus AB517709.1 DAM 5OWODM3 T, S, G, K, CEF, AM, SXT, AMC, SU bla T E M Serratia marcescens FJ607982.1 DAM 5OWODM3 T, AM, AMC, CEF, SU  bla S H V  Dam 6 f Acinetobacter baumannii JF919866.1 DAM 6OWIRW T, FF, S, C, AM, SXT, AMC, SU bla T E M Bacillus thuringiensis JN377782.1 DAM 6OWIRW SU, AM, T, SXT, RIF, LIN, CIP, GEN bla T E M bla S H V Klebsiellasp. DQ989215.2 DAM 6OWIRW T, S, AM, SXT, SU bla T E M  bla C T X Klebsiella pneumoniae CP002910.1 DAM 6OWIRW T, S, C, AM, SXT, AMC, SU bla T E M bla S H V Klebsiella oxytoca JF317350.1 DAM 6OWIRW K, AM, SXT, SU   bla C T X Morganella morganii AB089245.1 DAM 6OWIRW T, S, AM, AMC, SU   bla C T X Proteus vulgaris JN630888.1 DAM 6OWIRW T, FF, S, C, CEF, SXT, N, AMC, SU bla T E M Serratia marcescens JF429936.1 DAM 6OWIRW T, S, C, CEF, AM, AMC, SU  bla S H V Uncultured bacterium JN595068.1 DAM 6OWIRW S, G, K, AM, SU bla T E M Bacillus altitudinis HQ432811.1 DAM 6OWIFW SU, AM, T, E, SXT, RIF, LIN bla T E M bla S H V Alcaligenessp. JF707602.1 DAM 6OWIM1 T, S, G, K, CEF, AM, AMC, SU bla T E M Alcaligenes faecalis HM145896.1 DAM 6OWIM1 T, S, G, K, CEF, AM, AMC, SU bla T E M bla S H V Alcaligenessp. JF303893.1 DAM 6OWIM2 T, S, G, K, N, CEF, AM, SU  bla S H V Citrobacter freundii JN644567.1 DAM 6OWIM1 T, S, AM, SXT, N, AMC, SU bla T E M Klebsiella pneumoniae CP002910.1 DAM 6OWIM1 T, S, CEF, AM, SU  bla S H V Proteus mirabilis AB626123.1 DAM 6OWIM2 T, S, C, N, AMC, SXT  bla S H V Codes. aObafemi Awolowo University, Ife, Osun State; IRW = Ife raw water; IFFW = Ife treated water; IFM1 = Ife municipal tap 1; IFM2 = Ife municipal tap 2; bEde, Osun State; EDRW = Ede raw water; EDFW = Ede treated water; EDM1 = Ede municipal tap 1; EDM2 = Ede municipal tap 2; cAsejire, Oyo State, Nigeria; 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 2; fOwena-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 sequence. Ampicillin (AM); ceftiofur (CEF); chloramphenicol (C); florfenicol (FF); kanamycin (K); streptomycin (S); gentamycin (GEN); tetracycline (T); nalidixic acid (N); sulfamethoxazole (SU); sulfamethoxazole/trimethoprim (SXT); amoxicillin/clavulanic acid (AMC); erythromycin (E); rifampin (RIF); lincomycin (LIN); ciprofloxacin (CIP).


## 3.1. Bacteria Isolates

Isolates used in this study were selected from our previous studies [3–5] and represented α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Flavobacteriia, Bacilli, and Actinobacteria with 33, 66, and 68 being isolated from all treated, raw, and municipal taps, respectively (Table 1).Proteus was the most frequent (18.18%) isolated Gram-negative genus from the treated water whileKlebsiella was the most frequently (15.15%) isolated genus from raw water.Bacillus was the most common isolated Gram-positive genus for treated and municipal water.Table 1
Classes and families of selected bacteria.
Source Class Family Number (% of total from source) Raw water α-Proteobacteria Brucellaceae 1 (1.51) β-Proteobacteria Alcaligenaceae 9 (13.64) Neisseriaceae 1 (1.51) γ-Proteobacteria Enterobacteriaceae 27 (40.91) Moraxellaceae 2 (3.03) Aeromonadaceae 6 (9.09) Xanthomonadaceae 2 (3.03) Flavobacteriia Myroidaceae 1 (1.51) Uncultured bacteria clone  3 (4.55) Bacilli Bacillaceae 10 (15.15) Staphylococcaceae 3 (4.55) Actinobacteria Microbacteriaceae 1 (1.51) Total raw water  66  Treated water α-Proteobacteria Caulobacteraceae 1 (3.03) β-Proteobacteria Alcaligenaceae 5 (15.15) Neisseriaceae 1 (3.03) γ-Proteobacteria Enterobacteriaceae 10 (30.30) Flavobacteriia Myroidaceae 1 (3.03) Uncultured bacteria clone  2 (6.06) Bacilli Bacillaceae 12 (36.36) Staphylococcaceae 1 (3.03) Total treated water  33  Municipal taps α-Proteobacteria Caulobacteraceae 1 (1.47) β-Proteobacteria Alcaligenaceae 7 (10.29) Neisseriaceae 3 (4.41) γ-Proteobacteria Enterobacteriaceae 21 (30.88) Moraxellaceae 6 (8.82) Flavobacteriia Myroidaceae 3 (4.41) Uncultured bacteria clone Bacilli Bacillaceae 26 (38.24) Staphylococcaceae 1 (1.47) Total municipal tap  68 Note: identification was based on 16S rDNA sequencing. These bacteria were obtained from our previous works [3–5].


## 3.2. PCR-Positive Isolates

In this study, 61 isolates out of 164 MDR isolates were PCR-positive for at least one targeted gene. Highest occurrence ofbla gene among Gram-negative bacteria compared to Gram-positive bacteria was observed. Most commonly isolated genus carryingbla gene isKlebsiella (18.03%) followed byProteus spp. (14.75%). BlaTEM was detected in the majority of beta-lactam resistant isolates (50/61) while blaCTX was rarely detected (3/61) (Table 2). A combination of two genes, blaSHV + blaTEM or blaCTX + blaTEM, was observed in 11 and 1 bacteria, respectively (Table 2). Other genera, includingAquitalea,Comamonas,Enterobacter,Leucobacter,Lysinibacillus,Pantoea,Pseudochrobactrum,Sphingobacterium, andRalstonia, were tested but were PCR-negative for resistance genes.Table 2
Characterization of a number of cultured bacterial isolates encoding differentESBLgenotypes.
Genus/species/accession number Source Resistant phenotypes bla T E M bla S H V bla C T X Dam 1 a Escherichia coli AP010960.1 DAM 1IRW T, AM, S, C, N, SXT, SU bla T E M Uncultured bacterium clone JN595783.1 DAM 1IFFW T, FF, AM, G, SU bla T E M Bacillus thuringiensis JN377782.1 DAM 1IFFW SU, AM, T, E, SXT, RIF, LIN, GEN bla T E M Brevundimonas diminuta EU545397.1 DAM 1IFFW S, G, K, N, AM, SXT, SU  bla S H V Proteus mirabilis AB626123.1 DAM 1IFFW FF, T, S, G, K, C, AMC, AM, SU, SXT bla T E M Bacillus thuringiensis JN377782.1 DAM 1IFM1 SU, AM, E, SXT, RIF, LIN bla T E M bla S H V  Dam 2 b Bacillus altitudinis HQ432811.1 DAM 2EDRW SU, E, RIF, LIN, AM  bla S H V Bordetellasp. HQ840720.1 DAM 2EDRW T, FF, S, C, N, CEF, AM, SXT, SU bla T E M Proteus vulgaris JN630888.1 DAM 2EDRW T, AM, SXT, SU bla T E M Staphylococcussp. JN695710.1 DAM 2EDRW SU, T, E, SXT, RIF, LIN, AM bla T E M Stenotrophomonas maltophilia JN703732.1 DAM 2EDRW T, S, K, CEF, AM, AMC, SU bla T E M bla S H V Bacillus cereus AP007209.1 DAM 2EDFW SU, AM, T, E, SXT, RIF, LIN bla T E M Morganellasp. GQ179706.1 DAM 2EDFW T, S, AM, SXT, SU bla T E M Psychrobactersp. HQ730697.1 DAM 2EDM2 T, S, CEF, AM, SXT, SU bla T E M  Dam 3 c Alcaligenes faecalis JN162124.1 DAM 3ARW S, CEF, AM, SXT, SU  bla S H V Klebsiella pneumoniae AB675600.1 DAM 3ARW FF, T, S, C, AMC, CEF, AM, SU, SXT bla T E M Leucobacter komagatae AJ746337.1 DAM 3ARW T, S, AM, G, K, SXT, N, SU bla T E M Proteus mirabilis AB626123.1 DAM 3ARW T, S, AM, N, SXT, SU bla T E M Uncultured bacterium clone JN595783.1 DAM 3ARW T, G, K, C, N, CEF, AM, SXT, AMC, SU bla T E M Bacillus pumilus EF010673.1 DAM 3AFW SU, AM, T, E, SXT, RIF, LIN bla T E M Klebsiella pneumoniae JF919909.1 DAM 3AFW T, S, C, AM, SXT, SU  bla S H V Myroides odoratus AB517709.1 DAM 3AFW FF, T, S, G, K, C, AM, SXT, AMC, SU bla T E M Proteus vulgaris JN630888.1 DAM 3AFW FF, T, S, C, N, CEF, AM, SXT, AMC, SU bla T E M Acinetobacter calcoaceticus DAM 3AM2 S, AMC, AM, SU bla T E M bla S H V Chromobacteriumsp. AB426118.1 DAM 3AM1 T, S, CEF, AM, SXT, SU, AMC, SU bla T E M Klebsiella pneumoniae JF513171.1 DAM 3AM2 FF, C, CEF, AM, SXT, AMC, SU, AMC, SU bla T E M bla S H V  Dam 4 d Aeromonas caviae AB626132.1 DAM 4ERW T, S, AM, SXT, N, AMC, SU bla T E M Alcaligenes faecalis HQ161777.1 DAM 4ERW T, S, K, AM, SU bla T E M Alcaligenes faecalis JN162124.1 DAM 4ERW T, S, K, AM, SU bla T E M Klebsiella pneumoniae JN545039.1 DAM 4ERW S, CEF, AM, SXT, AMC, SU bla T E M Klebsiella pneumoniae JN545039.1 DAM 4ERW T, K, N, AM, SU bla T E M bla S H V Klebsiella pneumoniae JF513172.1 DAM 4ERW T, FF, S, C, AM, SXT, AMC, SU bla T E M bla S H V Morganella morganii FJ971868.1 DAM 4ERW T, S, K, CEF, AM, SXT, AMC, SU bla T E M Proteus vulgaris JN630888.1 DAM 4ERW T, C, CEF, AM bla T E M Proteus mirabilis GU420988.1 DAM 4ERW T, S, K, N, AM, SXT, AMC, SU bla T E M Proteus vulgaris JN630888.1 DAM 4ERW FF, T, S, G, K, C, AM, SXT, N, AMC, SU bla T E M Providencia vermicola NR_042415.1 DAM 4ERW T, G, AM, SU bla T E M Trabulsiella guamensis AB273737.1 DAM 4ERW T, C, CEF, AM, SU, SXT  bla S H V  Dam 5 e Klebsiellasp. JN036433.1 DAM 5OWODFW T, FF, S, C, AM, SXT, AMC, SU bla T E M bla S H V Alcaligenes faecalis JN162124.1 DAM 5OWODM2 T, S, G, K, C, AM, SXT, SU bla T E M Escherichia coli CP003034.1 DAM 5OWODM2 T, AM, AMC, SU bla T E M Escherichia coli CP003034.1 DAM 5OWODM2 T, AM, AMC, SU bla T E M Morganella morganii AM931264.1 DAM 5OWODM1 T, S, CEF, AM, SXT, AMC, SU bla T E M Morganella morganii AB089245.1 DAM 5OWODM3 T, S, K, CEF, SXT, AMC, SU bla T E M Myroides odoratus AB517709.1 DAM 5OWODM3 T, S, G, K, CEF, AM, SXT, AMC, SU bla T E M Serratia marcescens FJ607982.1 DAM 5OWODM3 T, AM, AMC, CEF, SU  bla S H V  Dam 6 f Acinetobacter baumannii JF919866.1 DAM 6OWIRW T, FF, S, C, AM, SXT, AMC, SU bla T E M Bacillus thuringiensis JN377782.1 DAM 6OWIRW SU, AM, T, SXT, RIF, LIN, CIP, GEN bla T E M bla S H V Klebsiellasp. DQ989215.2 DAM 6OWIRW T, S, AM, SXT, SU bla T E M  bla C T X Klebsiella pneumoniae CP002910.1 DAM 6OWIRW T, S, C, AM, SXT, AMC, SU bla T E M bla S H V Klebsiella oxytoca JF317350.1 DAM 6OWIRW K, AM, SXT, SU   bla C T X Morganella morganii AB089245.1 DAM 6OWIRW T, S, AM, AMC, SU   bla C T X Proteus vulgaris JN630888.1 DAM 6OWIRW T, FF, S, C, CEF, SXT, N, AMC, SU bla T E M Serratia marcescens JF429936.1 DAM 6OWIRW T, S, C, CEF, AM, AMC, SU  bla S H V Uncultured bacterium JN595068.1 DAM 6OWIRW S, G, K, AM, SU bla T E M Bacillus altitudinis HQ432811.1 DAM 6OWIFW SU, AM, T, E, SXT, RIF, LIN bla T E M bla S H V Alcaligenessp. JF707602.1 DAM 6OWIM1 T, S, G, K, CEF, AM, AMC, SU bla T E M Alcaligenes faecalis HM145896.1 DAM 6OWIM1 T, S, G, K, CEF, AM, AMC, SU bla T E M bla S H V Alcaligenessp. JF303893.1 DAM 6OWIM2 T, S, G, K, N, CEF, AM, SU  bla S H V Citrobacter freundii JN644567.1 DAM 6OWIM1 T, S, AM, SXT, N, AMC, SU bla T E M Klebsiella pneumoniae CP002910.1 DAM 6OWIM1 T, S, CEF, AM, SU  bla S H V Proteus mirabilis AB626123.1 DAM 6OWIM2 T, S, C, N, AMC, SXT  bla S H V Codes. aObafemi Awolowo University, Ife, Osun State; IRW = Ife raw water; IFFW = Ife treated water; IFM1 = Ife municipal tap 1; IFM2 = Ife municipal tap 2; bEde, Osun State; EDRW = Ede raw water; EDFW = Ede treated water; EDM1 = Ede municipal tap 1; EDM2 = Ede municipal tap 2; cAsejire, Oyo State, Nigeria; 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 2; fOwena-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 sequence. Ampicillin (AM); ceftiofur (CEF); chloramphenicol (C); florfenicol (FF); kanamycin (K); streptomycin (S); gentamycin (GEN); tetracycline (T); nalidixic acid (N); sulfamethoxazole (SU); sulfamethoxazole/trimethoprim (SXT); amoxicillin/clavulanic acid (AMC); erythromycin (E); rifampin (RIF); lincomycin (LIN); ciprofloxacin (CIP).


## 4. Discussion

The hazard associated with the pathogenicity of microbes is aggravated by its ability to resist destruction by antibiotics [2]. In this study, beta-lactamase producing bacteria and genes (i.e., blaCTX and blaTEM) were detected from every sampled water distribution system. This is similar to the report of Xi et al. [13] who also investigated the prevalence and dynamics of heterotrophic antibiotics resistance bacteria and genes in drinking water source and treated drinking water using culture-dependent methods and molecular techniques. The authors observed the presence of blaTEM and blaSHV genes in all water samples except one, which is evidence that these genes are distributed widely in drinking water systems. This is similar to what we also reported in Table 3, showing the spread of these beta-lactamase resistance genes among all raw, final, and municipal tap sources. The results showed that even among bacteria from the municipal tap and final treated water from the dam which are the point of consumer consumption blaTEM and blaCTX occurred among bacteria from these sources in high number. Xi et al. [13] also observed selective increases in the levels of both genes in tap water due to either water treatment or regrowth within drinking water distribution systems. This, as they therefore reported, suggested the spread of at least some beta-lactam-resistant determinants through drinking water distribution systems. However, in this study, it is important to point out that every site is different for numerous variables making it impossible to derive meaningful correlations between water treatment practices and the occurrence of beta-lactamase resistance genes. Given these differences, the only practical means to assess the effects of water treatment practices (which is not our goal with this paper) would be to test changes with experimental manipulation. We have been reluctant to provide significant detail of the sample sites precisely because we do not wish to imply that there are defendable correlations between occurrence and site characteristics, nor do we wish to encourage readers to draw such inferences.Table 3
Number of bacteria and genes observed from all raw, treated, and municipal taps carrying at least onebla gene tested for in this study.
Genus bla genes from raw water bla genes from final water bla genes from municipal taps Bacteria number bla T E M bla S H V bla C T X Bacteria number bla T E M bla S H V bla C T X Bacteria number bla T E M bla S H V bla C T X Acinetobacter spp. 1 1 0 0 0 0 0 0 1 1 1 0 Aeromonas spp. 1 1 0 0 0 0 0 0 0 0 0 0 Alcaligenes spp. 3 2 1 0 0 0 0 0 4 3 1 0 Bacillus spp. 2 1 2 0 4 4 1 0 1 1 1 0 Bordetella spp. 1 1 0 0 0 0 0 0 0 0 0 0 Brevundimonas spp. 0 0 0 0 1 0 1 0 0 0 0 0 Chromobacteriumspp. 0 0 0 0 0 0 0 0 1 1 0 0 Citrobacterspp. 0 0 0 0 0 0 0 0 1 1 0 0 E. coli 1 1 0 0 0 0 0 0 2 2 0 0 Klebsiella spp. 7 6 3 1 2 0 1 0 2 1 2 0 Leucobacter spp. 1 1 0 0 0 0 0 0 0 0 0 0 Morganella spp. 2 1 0 1 1 1 0 0 2 2 0 0 Myroides spp. 0 0 0 0 1 1 0 0 1 1 0 0 Proteus spp. 6 6 0 0 2 2 0 0 1 0 1 0 Providencia spp. 1 1 0 0 0 0 0 0 0 0 0 0 Psychrobacterspp. 0 0 0 0 0 0 0 0 1 1 0 0 Serratia spp. 1 0 1 0 0 0 0 0 1 0 1 0 Staphylococcus spp. 1 1 0 0 0 0 0 0 0 0 0 0 Stenotrophomonas spp. 1 1 1 0 0 0 0 0 0 0 0 0 Trabulsiella spp. 1 0 1 0 0 0 0 0 0 0 0 0 Uncultured bacteria clone 2 2 0 0 1 1 0 0 0 0 0 0  Total 32 26 9 2 12 9 3 0 18 14 7 0Moreover, we observed that there were beta-lactam resistant strains that were negative for the PCR assays used in this study. The most commonly detectedbla genes were blaTEM and blaSHV amongKlebsiella. This finding is contrary to previous reports [14–16]. They observed dominance of blaCTX among non-TEM and SHV bacteria from clinical environment which were similar to what Ojdana et al. [17] reported among clinical samples from Poland. Additionally, studies onPseudomonas spp. isolated from these water distribution systems also observed a higher occurrence of blaTEM (40.9%) and blaCTX (27.3%) while none of the pseudomonads showed the presence of blaCTX [18]. Our observation of the highest occurrence ofbla gene among Gram-negative bacteria, when compared to Gram-positive bacteria, in this study is similar to reports of [19, 20]. These authors also confirm blaTEM that was frequently detected among Gram-negative bacteria from this study as the most commonbla gene in their studies.In this study, environmental bacteria belonging to each of these generaBordetella,Brevundimonas,Chromobacterium,Providencia,Psychrobacter,Stenotrophomonas,Trabulsiella, andAeromonas possess at least one of the beta-lactamase resistance genes tested; the most common among them is blaTEM. Occurrence of this gene in these environmental isolates is contrary to the report that ESBL production is mostly found to occur among enteric species [21]. The firstbla genes (blaBOR-1 and blaOXA-2) were reported inBordetella by Kadlec et al. [22]. However, we did not come across any publication where blaTEM has been reported in this bacterium. This could be the first report of this gene in this bacterium. Nevertheless, blaTEM has been reported inProvidencia [23],Stenotrophomonas [24], andAeromonas [25]. In fact, anotherbla gene such as blaLI has been reported inStenotrophomonas from China [26] and blaSHV and blaCTX-M have been reported inAeromonas [25]. Moreover, no blaSHV has also been reported inTrabulsiella; this report probably might be its first description.The association of more than oneβ-lactamase within the same isolate has been reported [27, 28]. However, from our studies the most common of this association is blaSHV + blaTEM. This was detected amongAcinetobacter,Alcaligenes,Bacillus,Klebsiella, andStenotrophomonas while the combination of blaCTX and blaTEM was only observed inKlebsiella. This occurrence denotes the wider dissemination of thesebla genes probably due to involvement of genetic element in mobilization of these genes [29]. These same authors [29] also observed various combinations of blaCTX, blaTEM, and blaSHV inKlebsiella from clinical isolates in India.The occurrence of ESBL genes among bacteria from this study has a public health implication. Previous studies have shown that potential ESBL species such asK. pneumonia, the most frequent bacterium withbla gene in this study, andE. coli have a high tendency to possess and transferbla genes [30]. However, this may occur through conjugation because the genes are often found in mobile elements like transposons and integrons [31]. Some of these species may be pathogenic strains that have the potential to cause life-threatening diseases and widespread outbreak. For instance, Zhang et al. [32] have reported that blaCTX-M and blaTEM genes in opportunistically pathogenicKlebsiella spp. have been associated with nosocomial infections and outbreak of diarrhea. Therefore, occurrence of these bacteria especially in the drinking water poses a lot of danger to health, economy, and social well-being of consumers. This populace could also be exposed to these genes carrying pathogenic species in food and food products by the use of the contaminated water for domestic purposes, farming, and agriculture. It should also be noted that the fact that these species are multidrug resistance deepens the gravity of the situation. However, from our observation during sampling, the possible source of these MDR bacteria especially in the raw water could be from run-off from agricultural farmlands located very close to some of these constructed dams [4]. Some of these farmlands make use of organic fertilizers which may consist of unmetabolized antibiotics which may eventually get to the water through run-off, which may cause selective pressure on the bacteria in the aquatic systems.From the bacteria found in Nigeria, many studies have described the occurrence ofbla genes among clinical isolates. For example, Akujobi et al. [33] reported blaTEM inE. coli while Akinniyi et al. [34] reported the gene not only inE. coli but also amongKlebsiella,Salmonella,Citrobacter,Enterobacter,Pseudomonas, andProteus. The highest prevalence (5.6%) was also inKlebsiella which is similar to our findings. However, from environmental isolates few studies seem to have been conducted. Moreover, Adelowo et al. [35] reported blaTEM inE. coli from well water while Chikwendu et al. [36] described not only blaTEM amongPseudomonas from river and aquaculture samples but also blaSHV. Moreover, this study seems to be the first report describing these genes among a wide diversity of environmental bacteria from Nigeria drinking water distribution systems. It is therefore important to raise public and health worker awareness in terms of prevention of outbreak of MDR infectious pathogens among consumers. This also undermines the need for government agencies controlling these dams and health organizations to initiate measures to effectively control the release of contaminant into the environment. It would also be good to continually isolate bacteria from other water distribution systems in Nigeria and to carry out further molecular testing for the presence of otherblagenes, characterize, and determine whether these genes are present on transferable plasmids, transposons, or integrons, which would enhance easy spreading.


## 5. Conclusion

The occurrence of beta-lactamase producing bacteria and genes in all sampled water in this study, especially treated drinking water, showed that these water distribution systems could serve as a vehicle for transmission of these antibiotic resistance bacteria and genes to consumers, hence, of a great public health concern.



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*Source: 7149295-2016-08-03.xml*