Molecular and Cellular Microbiology / Biomedical Sciences | Clinical Microbiology and Biomedical Sciences
Microbiol. Biotechnol. Lett. 2020; 48(2): 230-235
https://doi.org/10.4014/mbl.1911.11008
Dickson Musa 1, Harun Aremu 2*, Abraham Ajayi 3 and Stella Smith 4
1Department of Biochemistry, IBB University, Lapai, Niger State, Nigeria, 2Department of Biochemistry, Osun State University, Osogbo, Osun State, Nigeria, 3Department of Microbiology, University of Lagos, Lagos, Nigeria, 4Molecular Biology and Biotechnology Department, Nigerian Institute of Medical Research Yaba, Lagos, Nigeria
The global evolution of antibiotic resistance has threatened the efficacy of available treatment options with ravaging impacts observed in developing countries. As a result, investigations into the prevalence of antibiotic resistance and the role of plasmids are crucial. In this study, we investigated the presence and distribution of blaTEM and gyrA genes, plasmid profiles, and the antimicrobial susceptibility pattern of Salmonella strains isolated from raw meat and stool sources across Niger State, Nigeria. Ninety-eight samples, comprising 72 raw meat and 26 stool samples, were screened for Salmonella spp. The antimicrobial susceptibility of Salmonella isolates to 10 commonly used antimicrobial agents was determined using the Kirby- Bauer disc diffusion method. Isolates were further analyzed for plasmids, in addition to PCR amplification of beta-lactamase (blaTEM) and gyrA genes. A total of 31 Salmonella spp. were isolated, with 22 from raw meat (70.97%) and 9 from stool (29.03%). Salmonella spp. with multiple resistance patterns to ceftazidime, cefuroxime, ceftriaxone, erythromycin, ampicillin, cloxacillin, and gentamicin were detected. Ofloxacin and ciprofloxacin were found to be the most effective among the antibiotics tested, with 67.7% and 93.5% susceptible isolates, respectively. Nine (29.03%) isolates harbored plasmids with molecular sizes ranging between 6557 bp and 23137 bp. PCR amplification of gyrA was detected in 1 (3.23%) of the 31 isolates while 28 isolates (90.32%) were positive for blaTEM. This study shows the incidence of antibiotic resistance in Salmonella isolates and the possible role of plasmids; it also highlights the prevalence of ampicillin resistance in this local population.
Keywords: Salmonella spp., plasmid, antibiotics resistance, resistance genes, polymerase chain reaction, Niger State
The global trend of resistance to antibacterial agents is alarming and poses grave threat. Consequently, this has prompted investigation into the incidence of antibiotics resistance in bacteria and underlying mechanisms involved in proliferation of resistance in various environments [4] in addition to the role of plasmid. Numerous ecological studies have shown that increased use of antibiotic in agriculture and treatment of human infections [5] contribute to the emergence of antibiotic resistance in various bacterial genera [6].
Several literatures have reported the roles of plasmid and resistance genes in antibiotics resistance [7, 8]. Bacterial antibiotics resistance can be intrinsic or acquired via horizontal gene transfer of genetic mobile elements associated with resistance and mutations causing changes in genetic composition [4]. From earlier studies, resistance to ampicillin and ciprofloxacin has been attributed different genetic determinant including
This study was conducted with approval of ethics committee of Niger State Hospitals Management Board of Ministry of Health, Niger State (NSHMB No. 2018-08-009).
This study was conducted across Niger State, Nigeria. Stool samples were collected from General Hospitals including Lapai, Bida, Minna, Suleja, Kontagora and Wushishi and raw meat samples were collected from sellers across the state. A total of ninety-eight (98) samples consisting of 26 stool samples collected from suspected typhoid fever patients and 72 raw meat samples from raw meat sellers. Table 1 shows distribution of samples across the state. Samples were aseptically collected in sterile sample bottles and Ziploc bags and transported to the laboratory at 4℃ for analysis.
Table 1 . Distribution of Samples across Niger State.
Sample | Bida | Lapai | Minna | Suleja | Kontangora | Wushishi | Total |
---|---|---|---|---|---|---|---|
Stool | 5(2) | 5(2) | 5(1) | 5(2) | 1(0) | 5(2) | 26(09) |
Raw meat | 12(6) | 12(5) | 12(3) | 12(3) | 12(4) | 12(1) | 72(22) |
*Values in “( )” indicate no. of
Meat (25 g) and stool (5 g) samples were enriched and placed in sterile selenite broth and incubated for 24 h at 37℃. The aliquots were cultured in
Antimicrobial susceptibility of
Extraction of plasmid was performed on isolates using TENS-Mini Prep as described by Liu [14]. A 1% agarose gel was used for plasmid horizontal electrophoresis. Hind III digest of Lambda phage was used as a standard molecular marker.
DNA extraction was carried out using the Qiagen QIAmp mini DNA kit (Germany) according to the manufacturers’ specification.
Targeting primers shown in Table 2, a 20 μl PCR reaction was carried out. The reaction mixture contained a buffer of 1x hot FirePol Master Mix (Solis BioDyne; Estonia), dNTPs (200 μM), each primer (20 pmol), hot DNA polymerase (2 unit), magnesium chloride (2 mM), proofreading enzyme and sterile distilled water (final volume 20 μl). Amplification reactions were performed under the following conditions:
Among the 98 samples (72 meat and 26 stool),
As shown in Table 3,
Table 3 . Antibiotic susceptibility pattern of bacteria isolated from stool and raw meat samples (n = 31).
Antibiotic | Resistant (n) | Intermediate (n) | Susceptibility (n) |
---|---|---|---|
Ceftazidime | 31 | - | - |
Cefuroxime | 28 | 1 | 2 |
Ciprofloxacin | 1 | 1 | 29 |
Gentamicin | 21 | 2 | 8 |
Ceftriaxone | 31 | - | - |
Erythromycin | 24 | 5 | 2 |
Ampicillin | 31 | - | - |
Cloxacillin | 30 | - | 1 |
Ofloxacin | 9 | 1 | 21 |
The PCR detection of resistance genes was carried out as observed in Fig. 1, 2. Of the 31 isolates screened for
Plasmids are major vectors in the global spread of antibiotic resistance genes especially in gram-negative bacteria [8]. In the study, plasmids were detected in 9 (29.03%)
Furthermore, due to the rapid emergence of resistance to recommended therapeutic regimen (usually ampicillin and other third-generation cephalosporins or fluoroquinolones) for the treatment of
Similarly, fluoroquinolones resistance in
In conclusion, the development of antibiotics resistance continues to affect readily available treatment options which further burden the management of infectious disease in developing countries. Given the findings, we conclude there is an extensive spread of
The authors would like to acknowledge the valuable contributions of staff at the Molecular Biology and Biotechnology Department, Nigerian Institute of Medical Research, Yaba for their support during this research and AvH Foundation, Germany for the donation of photodocumentation system used in this study.
The authors have no financial conflicts of interest to declare.
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