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

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 include bla TEM, bla SHV, and bla CTX 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 bla TEM being the most abundant (50/61) and bla CTX being detected least (3/61). Klebsiella was the most frequently identified genus (18.03%) to harbour ESBL gene followed by Proteus (14.75%). Moreover, combinations of two ESBL genes, bla SHV + bla TEM or bla CTX + bla TEM, were observed in 11 and 1 isolate, respectively. In conclusion, classic bla TEM 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.


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 middleincome 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][3][4][5].
Arguably, the most clinically important antibiotic resistance genes are those that encode enzymes that hydrolyzelactams (bla genes) [6]. These traits confer high level resistance to -lactam antibiotics, which are the most widely used antibiotics in clinical and veterinary practice [7,8]. Extended Spectrum -Lactamase (ESBL) is group of enzymes that can hydrolyze a variety of -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 encoding -lactamase activity have been described in clinical environments including CTX-M , GES , HER , OXA , OXY , SED , SHV , SPM , VEB , VIM , and ampC [10]. Among the most common bla genes is the TEM-1 gene, the first described bla gene and a representative of the TEM group that now consists of more than 220 different distinct variants ("alleles"), which encode different amino acid polymorphisms that extend their 2 BioMed Research International 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][4][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][4][5] for the presence of selected beta-lactamase resistance genes using PCR.

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][4][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][4][5]. Molecular characterization of bacteria using 16S rDNA sequencing was determined as described in Adesoji et al. [11].

Bacteria Isolates.
Isolates used in this study were selected from our previous studies [3][4][5] and represented -Proteobacteria, -Proteobacteria, -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 while Klebsiella 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.

PCR-Positive Isolates.
In this study, 61 isolates out of 164 MDR isolates were PCR-positive for at least one targeted gene. Highest occurrence of bla gene among Gramnegative bacteria compared to Gram-positive bacteria was observed. Most commonly isolated genus carrying bla gene is Klebsiella (18.03%) followed by Proteus spp. (14.75%).

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., CTX and TEM ) 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 TEM and SHV 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 Note: identification was based on 16S rDNA sequencing. These bacteria were obtained from our previous works [3][4][5].
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 TEM and CTX 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.
Moreover, we observed that there were beta-lactam resistant strains that were negative for the PCR assays used in this study. The most commonly detected bla genes were TEM and SHV among Klebsiella. This finding is contrary to previous reports [14][15][16]. They observed dominance of CTX 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 on Pseudomonas spp. isolated from these water distribution systems also observed a higher occurrence of TEM (40.9%) and CTX (27.3%) while none of the pseudomonads showed the presence of CTX [18]. Our observation of the highest occurrence of bla 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 TEM that was frequently detected among Gram-negative bacteria from this study as the most common bla gene in their studies. In this study, environmental bacteria belonging to each of these genera Bordetella, Brevundimonas, Chromobacterium, Providencia, Psychrobacter, Stenotrophomonas, Trabulsiella, and Aeromonas possess at least one of the beta-lactamase resistance genes tested; the most common among them is TEM . 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 first bla genes ( BOR-1 and OXA-2 ) were reported in Bordetella by Kadlec et al. [22]. However, we did not come across any publication where TEM has been reported in this bacterium. This could be the first report of this gene in this bacterium. Nevertheless, TEM has been reported in Providencia [23], Stenotrophomonas [24], and Aeromonas [25]. In fact, another bla gene such as LI has been reported in Stenotrophomonas from China [26] and SHV and CTX-M have been reported in Aeromonas [25]. Moreover, no SHV has also been reported in Trabulsiella; this report probably might be its first description.
The association of more than one -lactamase within the same isolate has been reported [27,28]. However, from our studies the most common of this association is SHV + TEM . This was detected among Acinetobacter, Alcaligenes, Bacillus, Klebsiella, and Stenotrophomonas while the combination of CTX and TEM was only observed in Klebsiella. This occurrence denotes the wider dissemination of these bla genes probably due to involvement of genetic element in mobilization of these genes [29]. These same authors [29] also observed various combinations of CTX , TEM , and SHV in Klebsiella 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 as K. pneumonia, the most frequent bacterium with bla gene in this study, and E. coli have a high tendency to possess and transfer bla 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 lifethreatening diseases and widespread outbreak. For instance, Zhang et al. [32] have reported that CTX-M and TEM genes in opportunistically pathogenic Klebsiella 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 runoff 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 of bla genes among clinical isolates. For example, Akujobi et al. [33] reported TEM in E. coli while Akinniyi et al. [34] reported the gene not only in E. coli but also among Klebsiella, Salmonella, Citrobacter, Enterobacter, Pseudomonas, and Proteus. The highest prevalence (5.6%) was also in Klebsiella which is similar to our findings. However, from environmental isolates few studies seem to have been conducted. Moreover, Adelowo et al. [35] reported TEM in E. coli from well water while Chikwendu et al. [36] described not only TEM among Pseudomonas from river and aquaculture samples but also SHV . 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 other bla genes, characterize, and determine whether these genes are present on transferable plasmids, transposons, or integrons, which would enhance easy spreading.

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.