PCR Analysis of Resistant Bacteria Strains Isolated from River Sokoto, Northwestern Nigeria

Thirteen resistant bacteria strains from River Sokoto namely Salmonella typhi, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis, Enterobacter cloacae, Klebsiella oxytoca, Staphylococcus saprophyticus, Providencia rettgeri, Klebsiella pneumonae and Enterobacter aerogenes recovered on Mueller-Hinton agar by disc diffusion method were subjected to PCR (Polymerase chain reaction) analysis to determine their antibiotic resistance genes. Forward and reverse copies of five primers (TEM, spvC, SHV, aacC3 and qnrS) were used in the PCR analysis. Aminoglycoside resistance genes (aacC3) were detected in the majority of the isolates such as E. coli, Shigella flexneri, Enterobacter cloacae, Staphylococcus saprophytica and Enterobacter aerogenes with plasmid numbers ranging from 1 to 4 and molecular weights ranging from 185 bp to >10,200 bp. Virulence resistance genes (spvC) were detected in Salmonella typhi on two plasmids with molecular weights of 571 bp and >10,200 bp while quinolones resistance genes were detected on plasmid numbers ranging from 1 to 3 and molecular weights ranging from 400 bp to 1000 bp in Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis and Providencia rettgeri. Three different resistance genes namely β-lactam (blaTEM), virulence (spvC) and quinolones (qnrS) on 6 plasmids with molecular weights ranging from 428 bp to 1,200 bp were found in Pseudomonas aeruginosa while two resistance genes (aacC3 and qnrS) on 2 plasmids with molecular weights of 185bp and 428bp were detected in Klebsiella pneumoneae. Non-specific resistance genes alongside with specific resistance genes were however detected in the majority of the isolates. The study revealed that the resistance genes exhibited by resistant bacteria isolates from River Sokoto were mainly virulence, aminoglycoside and quinolones resistant genes. The scientists are therefore challenged on the need for development of new antibiotics to combat the infections caused by these resistant strains.


INTRODUCTION
Antibiotic resistance represents a global health problem, requiring better understanding of the ecology of antibiotic resistance genes (ARG), their selection and their spread in the environment (Marti et al., 2013). The overuse of antibiotics in human medicine and agriculture is a growing concern for public health. Overuse combined with inadequate wastewater treatment has led to the presence of antibiotic resistant bacteria and genes encoding antibiotic resistance in surface waters, river sediments, and the faeces of wild animals exposed to waste residuals. The multiple resistance of bacteria against antibiotics resulted from the fact that domestic and industrial waste were emitted into threw away without clarifying and so affect the underground water resources (Alzahrani and Gherbawy, 2011). The presence of antibiotic resistant bacteria in water sources throughout the world has been documented (Hamner, 2006;Baquero et al., 2008;Oyedeji, et al., 2011). Exposure to antibiotic resistant bacteria and the corresponding antibiotic resistance genes (ARG) in surface water can occur when the waters are used for drinking or recreation (Alzahrani and Gherbawy, 2011). Infection by antibiotic-resistant strains of pathogenic bacteria can have severe health implications for the sickened individual including more virulent strains and less treatment options (Barza and Travers, 2002).
In Sokoto, Nigeria, scarcely or seldom investigation was done on the assessment of pathogenic bacterial strains in River water using molecular techniques. In this study, we therefore examined genotypic characteristics of antibiotic resistant bacteria isolates recovered from six sampling points on River Sokoto. Bacterial cell preparation Bacterial cells were prepared using the method described by Dubey (2009). Single colonies were picked from freshly streaked isolates on MacConkey plate and incubated into 5ml Luria Bertani (LB) broth medium and incubated overnight at 37 o C for 18 -24 hours. Young cells were then harvested in eppendorff tube by centrifugation at 4 o C, 8000rpm (6800xg) in a refrigerated micro-centrifuge for 30 seconds. The supernatants were decanted and cells harvested.

DNA extraction
DNA extraction of the bacterial cells was carried out by using a DNA extraction kit (QIAamp DNA mini kit; Qiagen K.K., Tokyo, Japan). Heating block was first set at 60 o C before starting the extraction. Into a 2ml tube was added 200µl of the bacterial cells in liquid medium. Four hundred microliter (400µl) of lyses buffer and 10µl proteinase K were added to the sample and the tube was placed on heat block at 60 o C for minimum of 1 hour. Four hundred microliter (400 µl) of phenol chloroform (1:1) was added to the lysate and vortexed briefly. The mixture was spinned at 10000rpm for 10 minutes to separate the phases. The upper layer was carefully removed with a pipette without taking the white interphase which contained the DNA. For the second time, 400µl of chloroform was added to the remainder and vortexed briefly. The mixture was spinned again at 10000rpm for 10 minutes to separate the phases and the upper layer was carefully removed without taking the white interphase. To the remainder, was added 400µl of 100% ethanol and 20µl of 3M sodium acetate. This was mixed by inverting the tube several times and the tube was incubated at -20 o C overnight. On the following day, the tube was spinned at maximum speed for 10 -30 minutes in refrigerated centrifuge and the ethanol was removed. Four hundred microliter (400µl) of 70% cold ethanol was added and spinned at maximum speed for 5 minutes at 4 o C to precipitate the DNA because DNA is not miscible in alcohol. This step was repeated again to get more DNA precipitated and the salt totally removed. All traces of ethanol was removed by spinning the tube for 30 seconds at high speed and the DNA was dry out by leaving the tube open for 3 -10 minutes. The pellet was re-suspended in 50µl sterile water for further analysis. Presence of DNA was confirmed by electrophoresing on an agarose gel containing ethidium bromide dye and checking under UV light.

Polymerase chain reaction
Amplification of resistant DNA fragments was performed using Applied Biosystems Taq DNA polymerase (Courtaboeuf, France) which is a Multiplex PCR kit. For reaction set-up, the PCR tube was placed on an ice pack and the following items were added to each isolate for single reaction: (a) Taq polymerase master mix (b) primers (forward and reverse) (c) template DNA and (d) distilled water. Five primers with known molecular weights were used to amplify DNAs from thirteen bacterial isolates to determine which bacteria gave PCR amplicons of the same size as the primer. Primers used in the multiplex PCR are listed in Table 1 below. The setup was loaded on a PCR machine, PTC-100, Programmable Thermal Controller (M J. Research, Inc., USA). Thermocycling conditions for 35 cycles were as follows: pre-denaturation at 95 o C for 5 minutes; denaturation at 95 o C for 30 seconds; optimized annealing temperature for 30 seconds at 52 o C; extension for 1 minute at 72 o C followed by a final extension at 72 o C for 5 minutes. PCR products were resolved by gel electrophoresis. The process was repeated for positive isolates.

Agarose gel electrophoresis
To confirm that DNA has actually been extracted, the DNA suspensions were subjected to agarose gel electrophoresis as follows: One percent (1%) agarose gel was prepared by dissolving 1g of agarose powder in 2ml of ten times concentration of Tris acetate ethylene diamine tetra acetate (TAE) buffer and 98ml distilled water in a 250ml beaker. This was heated in a microwave to dissolve the agarose. The gel was stained with 20µl of 5ml ethidium bromide dye and mixed. The gel was casted onto a gel electrophoresis tank with the combs inserted at the red bands to ensure easy view of the well and was allowed for 30 minutes to solidify. The casting combs were removed and 5µl of gel tracking dye (bromophenol blue) plus 15µl of the PCR products (making 20µl) were gently mixed and loaded into the well with the standard (Bioneer, 100 bp Plus DNA Ladder, Alameda, USA). The electrophoresis tank was covered and the electrodes connected to the power source for separation of sample based on molecular weight from cathode (-) to anode (+) at 100 mV for 45 minutes. At the completion of the electrophoresis, the gel was removed and viewed under a trans-illumination UV light (Gel Doc, Italy). The band pattern of the DNA fragments was then pictured and documented.

RESULTS
Results of molecular analysis of thirteen (13) resistant bacterial strains isolated from River Sokoto are presented in Fig. 1 as shown below:    Providencia rettgeri (lane 11). Three different resistance genes which are β-lactam (bla TEM ), virulence (spvC) and quinolones (qnrS) were found in Pseudomonas aeruginosa (lane 4) while two resistance genes (aacC3 and qnrS) were detected in Klebsiella pneumoneae (lane 12). However, non-specific resistance genes were detected in the majority of the isolates.

DISCUSSION
Plasmids harbor genes coding for specific functions including virulence factors and antibiotic resistance that permit bacteria to survive the hostile environment found in the host and resist treatment (Ramirez et al., 2014). The uncontrolled use of antibiotics for empirical treatment of infectious diseases has been implicated as a cause of high prevalence of these antibiotics resistance (Olofsson, 2006). According to Odeyemi et al. (2015), another way by which these isolated bacteria can develop resistance to the various antibiotics is through the transfer of antibiotic resistant gene from one organism to another through their plasmids. This assertion was in agreement with the findings of this study as similar antibiotic resistance genes were detected in different plasmid carrying bacteria organisms which heightened the risk of bacterial infections in the study area as a result of wide spread of resistant strains to various commonly used antibiotics. In this study, Salmonella virulence plasmid genes (spvC) were detected in the Salmonella typhi isolates analysed. These virulence genes must have been the determinant genes for the resistance earlier observed on Salmonella typhi isolates in the antibiotic sensitivity test. Salmonella plasmid virulence genes have been detected in various Salmonella species (Carlson et al., 1999;Ziemer and Steadham, 2003;Carattoli, 2003

CONCLUSION
This study revealed that the antibiotic resistance genes in resistant bacteria pathogens isolated from River Sokoto were mainly virulence, aminoglycoside and quinolones resistance genes. This occurrence will heighten the risk of bacterial infections and promote failure to antibiotic treatments in the study area as a result of wide spread of resistant strains to various commonly used antibiotics. People in the community should therefore be counseled on the usage and dosage of antibiotics.
Scientists are also challenged on the need for development of new antibiotics to combat the infections caused by these resistant bacteria.