Sudan Journal of Medical Sciences Volume 13, Issue no. 3, DOI 10.18502/sjms.v13i3.2955 Production and Hosting by Knowledge E Research Article Identification of Proteus mirabilis on Banknotes Using 16s rRNA Gene in Khartoum State Alaa Abdalla Mukhtar1, Noha Ahmed Abd Alfadil2, Malik Suliman Mohamed3, Hisham N Altayb4, Salaheldein G Elzaki5, and Mohamed Salih Hassan6 1Department of Pharmaceutical chemistry, Faculty of Pharmacy, Omdurman Islamic University, Khartoum, Sudan 2Department of pharmaceutics, Faculty of Pharmacy, University of Alneileen, Khartoum, Sudan 3Department of pharmaceutics, Faculty of Pharmacy, University of Khartoum, Khartoum, Sudan 4Department of microbiology, Faculty of Medical Laboratories, Sudan University of Science and Technology, Khartoum, Sudan 5Department of Epidemiology, Tropical Medicine Research Institute, National Centre for Research, Khartoum, Sudan 6Applied Bioinformatics Center, Africa City of Technology, Khartoum, Sudan Abstract Background: The presence of pathogenic bacteria in circulated currency was recorded as a public health hazard. In this study, all examined Sudanese banknotes (100%) were found to be contaminated by gram-negative bacteria. Proteus mirabilis were recovered from 10 examined notes (22.2%, f = 10), E. coli (13.3%, f = 6) and Klebsiella spp. (8.9%, f = 4) were also identified. Only the most resistant P. mirabilis isolate was identified using culture-based and 16S rRNA gene sequencing techniques. Methods: Proteus isolates were identified phenotypically and tested for their susceptibility to 16 of commonly used antibiotics, then most resistant isolate was confirmed genotypically via 16S rRNA gene amplification and sequencing. Bioinformatics analysis using BLAST for sequence similarity search, Clustal W program for multiple sequence alignment, MEGA7 software for phylogenetic analysis. Tree was constructed to show the evolutionary relationships of the obtained sequence with similar sequences in the databases using. Results: The obtained sequence was found to be 100% identical to P. mirabilis 16S rRNA gene using BLAST. The phylogenetic tree was constructed to show the evolutionary relationships of the obtained sequence with similar sequences in the databases using MEGA7 software, and the closest strain was found to be P. mirabilis strain from India (EU411047). Conclusion: This study has shown that some currency notes circulated at Khartoum transportation are carriers of antimicrobial-resistant P. mirabilis that could be potential source for their transmission in public. Keywords: Proteus mirabilis, banknotes, 16S rRNA, Khartoum State How to cite this article: Alaa Abdalla Mukhtar, Noha Ahmed Abd Alfadil, Malik Suliman Mohamed, Hisham N Altayb, Salaheldein G Elzaki, and Mohamed Salih Hassan (2018) “Identification of Proteus mirabilis on Banknotes Using 16s rRNA Gene in Khartoum State,” Sudan Journal of Medical Sciences, vol. 13, issue no. 3, pages 175–186. DOI 10.18502/sjms.v13i3.2955 Page 175 Corresponding Author: Alaa Abdalla Mukhtar; email: pharma199322@gmail.com Received 2 August 2018 Accepted 15 September 2018 Published 24 September 2018 Production and Hosting by Knowledge E Alaa Abdalla Mukhtar et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Editor-in-Chief: Prof. Mohammad A. M. Ibnouf http://www.knowledgee.com mailto:pharma199322@gmail.com https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ Sudan Journal of Medical Sciences Alaa Abdalla Mukhtar et al 1. Introduction Proteus mirabilis causes 90% of Proteus infections [1]. It has been implicated in menin- gitis, empyema, osteomyelitis, and gastroenteritis. Also, it frequently causes health care-associated infections of the urinary tract, surgical wounds and lower respiratory tract [2]. Identification of bacteria is frequently performed by isolation of the organ- isms and study of their phenotypic characteristics, including gram staining, morphol- ogy, culture requirements, and biochemical reactions [3]. However, these traditional techniques have some disadvantages. Firstly, they are time-consuming and laborious. Secondly, a variability of culture due to different environmental conditions may lead to ambiguous results. Thirdly, a pure culture is required to undertake identification mak- ing the identification of fastidious and unculturable bacteria difficult and sometimes impossible [4]. Moreover, phenotypic systems cannot account for the variable char- acteristics observed among members of the same species, resulting in poor precision upon repeated testing [5]. Construction of phylogeny tree based on phenotypic meth- ods may be more difficult because it needs a comparison of a large set of independent co-varying characters, and it is difficult to perform cladistic analyses based on it [6]. In addition to the fact that phenotype represents a very small part of each organism’s genome [7]. 16S rRNA gene is highly conserved within species and among species of the same genus, and hence, can be used as an alternative technique for identification of bacteria to the species level [8, 9]. Specific properties of the 16S rRNA gene include its ubiquitous distribution, mosaic structure [10], and relative stability that qualify it to be used in the taxonomic assign- ment and phylogenetic relationship determination [11]. The genotypic bacterial identi- fication begins with nucleotide sequence analysis of the PCR product of specific gene/s followed by a comparison of these sequences with known sequences stored in a database [12, 13]. The 16S rRNA gene is suitable for identification since its size (1500 bp) is large enough for bioinformatics purposes [14]. Direct sequencing of 16S rRNA genes from environmental samples has become a standard and convenient method of assessing microbial population abundance [15], structure, and function in microbial communities [16, 17]. This study aimed to identify the bacterial isolates recovered from Sudanese ban- knotes using culture-based and 16S rRNA gene sequencing techniques. DOI 10.18502/sjms.v13i3.2955 Page 176 Sudan Journal of Medical Sciences Alaa Abdalla Mukhtar et al 2. Materials and Methods 2.1. Sources of banknotes Prospective study was conducted in March 2016 in which a total of 45 Sudanese ban- knotes were randomly collected from different sources; Hospitals (f = 15), food sellers (f = 15), transporters (f = 15). Five ‘mint’ brand new notes were collected from the bank before being touched with bankers to be used as controls. These new banknotes were included in the study to ensure whether the banknotes are contaminated from their source or during handling in the circulation. The banknotes studied were two, five, ten, twenty and fifty Sudanese pounds. All banknotes were in good shape and not damaged and transported into sterile plastic petri dishes to the microbiological laboratory for bacterial isolation, identification and antibiotic sensitivity testing. 2.2. Phenotypic analysis of bacteria from Sudanese Banknotes 2.2.1. Bacterial extraction and biochemical tests The banknotes were moistened with sterile distilled water, swabbed both sides by cotton-tipped swab and directly inoculated on 5% blood agar and MaCconkey agar plates. For bacterial growth observation, the inoculated plates were incubated aero- bically at 37ºC for 24 hrs. The cultural characteristic of the recovered contaminants of each banknote were examined and the suspected colonies were stained by gram- staining method. The gram-negative, rod-shaped bacilli and swarm colonies on media were sub-cultured in nutrient agar plates for further identification tests. Biochemical tests (catalase test, oxidase test, motility, indole test, urease, glucose fermentation test and lactose fermentation test) were carried out [17]. 2.2.2. Susceptibility tests Samples were tested for their susceptibility to these antibiotics: Amoxicillin (AMX) 25 µg, Amoxyclav (AMC) 30 µg, Cephalexin (CN) 30 µg, Cefuroxime (CXM) 30 µg, Ceftri- axone (CTR) 30 µg, Ceftazidime (CAZ) 30 µg, Gentamicin (GEN) 10 µg, Kanamycin (K) 30 µg, Co-trimoxazole (COT) 25 µg, Erythromycin (E) 5 µg, Azithromycin (AZM) 15 µg, Ciprofloxacin (CIP) 30 µg, Levofloxacin (LE) 5 µg, Nitrofurantoin (NIT) 200 µg, Chlo- ramphenicol (C) 30 µg, and Meropenem (MEM) 10 µg using Kirby–Bauer disc diffusion method [18]. DOI 10.18502/sjms.v13i3.2955 Page 177 Sudan Journal of Medical Sciences Alaa Abdalla Mukhtar et al 2.3. Genotypic analysis of bacteria from Sudanese Banknotes 2.3.1. DNA extraction More resistant isolate recovered from examined Sudanese banknotes was subjected to genotypic analysis. Three colonies from pure sub-cultured isolate was suspended in 200µl 1x Phosphate Buffer Saline (PBS). Genomic DNA was extracted using chelex extraction protocol [19] and quantified using (GeneQuant, Amersham) according to manufacturer’s protocol. 2.3.2. 16S rRNA gene amplification Forward primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer 1495R (5′- CTACGGCTACCTTGTTACGA-3′) [20] were used to amplify 16S rRNA gene. The PCR reac- tion mixture (iTRON, Korea) were as follows: 2.5 unit Taq DNA polymerase, 2.5 mMmm dNTP, 1x reaction Buffer (10x), 1x Gel loading buffer, 5µl of template DNA, 1µL of primer (27F:10pmol/µ), 1µl of primer (1495R10 pmol/µl) and 15µl of distilled water mixed in a final volume of 25µl. The PCR conditions were; 94ºC for 5 minute, 37 cycles of 94ºC for 1 minute, 58ºC for 1 minute, 72ºC for 1 minute, and final extension at 72ºC for 10 minutes. Amplification was done using thermocycler system (BIO-RAD, USA). 2.3.3. Detection of amplified product by agarose gel electrophoresis The PCR products were assessed by gel electrophoresis. One percent agarose gel with 0.5% ethidium bromide were mixed and 5μl of PCR product and ladder (iTRON, Korea) were transferred into separated wells in the gel. The electric current was allowed at 100 volts for 30 minute, while UV Trans-illuminator (UVP, USA) was used for the observation of DNA bands [21]. The obtained fragment was sent to be sequenced in Microgen company (Seoul, South Korea). 2.4. Bioinformatics analysis The 16S rRNA gene sequence was analyzed using BLAST [22] with non-redundant (nr) NCBI GenBank database to find closely related bacterial 16S rRNA gene sequences. The 16S rRNA gene sequence of Proteus isolate was submitted to NCBI database with accession number (KY 039269). Nineteen sequences of high-quality 16S rRNA gene were selected from SILVA database [23] and subjected for multiple sequence alignment DOI 10.18502/sjms.v13i3.2955 Page 178 Sudan Journal of Medical Sciences Alaa Abdalla Mukhtar et al using Clustal W program [24] within BioEdit software [25], with total alignment score 1661794. The phylogenetic tree was constructed according to maximum likelihood method using MEGA 7 software [26]. 3. Results 3.1. Results of susceptibility tests All P. mirabilis isolates were completely sensitive to Meropenem (MEM), Levofloxacin (LEV), Ciprofloxacin (CIP), Gentamicin (GEN), Ceftazidime (CAZ), Ceftriaxone (CTR), Cephalexin (CN), and Amoxyclav (AMC), as well as they were completely resistant to Azithromycin (AZM) and Erythromycin (E). However, resistant strains of P. mirabilis isolates to Kanamycin (K), Chloramphenicol (C), Nitrofurantoin (NIT), Co-trimoxazole (COT), Cefuroxime (CXM) and Amoxicillin (AMX) 25µg were also detected, as shown in Table 1. T 1: Susceptibility test of P. mirabilis isolates using disc diffusion assay. antibiotics MEM C LEV CIP K GEN NIT COT AZM E CAZ CTR CXM CN AMC AMX Sensitive isolates 100% 86% 100% 100% 57% 100% 0% 43% 0% 100% 0% 100% 57% 100% 100% 57% Resistant isolates 0% 14% 0% 0% 14% 0% 57% 57% 100% 100% 0% 0% 43% 0% 0% 43% Intermedia te isolates 0% 0% 0% 0% 29% 0% 43% 0% 0% 0% 0% 0% 0% 0% 0% 0% Table 1 shows the results of susceptibility test of P. mirabilis isolates toward com- monly used antibiotics using disc diffusion assay. 3.2. DNA quantification results T 2: DNA concentrations in µg/l using GeneQuant. Sample No. Protein µg/l Ratio ssDNA µg/l Purity % Sample 1 0.4 1.2 39.0 69 Sample 2 0.3 1.4 39.5 75 Sample 3 0.4 1.3 48.0 72 Sample 4 0.3 1.3 38.8 74 Sample 5 0.3 1.5 52.0 82 Table 2 illustrates the DNA concentrations of five samples using GeneQuant machine. Only Sample 5 was adopted for further processes. DOI 10.18502/sjms.v13i3.2955 Page 179 Sudan Journal of Medical Sciences Alaa Abdalla Mukhtar et al 3.3. Fragments separation using gel electrophoresis Figure 1: Amplified fragment detection, L: ladder (500 bp), S: Sample 5. Figure 1 represents separated band of Sample 5 compared with bands of ladder using gel electrophoresis. 3.4. Multiple sequence alignment Figure 2: Multiple sequence alignment of 19 databases 16S rRNA genes with 16S rRNA gene of P. mirabilis isolate (KY 039269) from Sudan. Figure 2 shows the result of multiple sequence alignment of 19 databases 16S rRNA genes with 16S rRNA gene of P. mirabilis isolate (KY 039269) from Sudan. 3.5. Phylogenetic analysis Evolutionary history was inferred using maximum likelihood method based on Tamura- Nei model. The tree was constructed by MEGA7 software [26]. 16S rRNA gene of P. mirabilis isolate from Sudanese notes (KY 039269) is closely related to16S rRNA gene of P. mirabilis isolate from India (EU411047) as shown in Figure 3. DOI 10.18502/sjms.v13i3.2955 Page 180 Sudan Journal of Medical Sciences Alaa Abdalla Mukhtar et al Figure 3: Phylogenetic tree of 16S rRNA gene of P. mirabilis isolate from Sudanese notes (KY 039269). 4. Discussion The result of this study represents that 22.2% of tested Sudanese banknotes were contaminated by P. mirabilis that is agreed with results from India and Nigeria where it was found in banknotes at low frequency [27, 28]. Banknotes contamination rate is incredibly high at Khartoum transport circulation that may be due to more fre- quent exchange, poor hygiene and environmental conditions such as temperature and humidity [29]. The risk of transmission of pathogenic microorganisms and diseases by banknotes was reported worldwide, but most of these studies were performed in the tropical or sub-tropical regions of the world [30, 31]. Banknotes and coins were reported as carriers of potentially pathogenic microorganism since the beginning of the seventies [32, 33]. The 16S rRNA gene that is commonly used for identification and classification of microbes from environmental samples [34, 35] was used in this study for identification of banknotes isolates after biochemical tests. Banknotes-associated microorganisms detected previously using 16S rRNA gene [36, 37]. It can be used for identification for bacterial strains more accurately than it is possible with phenotypic analysis, allowing identification of strains that are poorly culturable or do not exhibit distinguishable phe- notypic traits [12]. In this study, the most resistant isolate toward examined antibiotics was chosen for DNA extraction and sequencing for identification and also to detect changes in 16S rRNA sequence that may explain their resistance to these antibiotics [38]. After quantification of extracted DNA, gel electrophoresis and DNA sequencing, DOI 10.18502/sjms.v13i3.2955 Page 181 Sudan Journal of Medical Sciences Alaa Abdalla Mukhtar et al the obtained 756 pb sequence with the aid of BLAST and multiple sequence align- ment confirmed that the isolate was P. mirabilis with 100% identity to the database sequence. It is known that the full-length or near-full-length 16S rRNA gene sequences is crucial for making confident genus and species-level taxonomic placements [39]. The 16S rRNA gene could be used as phylogenetic marker because of its functional constancy and the presence of conserved and variable sequence regions evolving at very different rates. It is also critical for the concurrent universal amplification and measurement of both close and distant phylogenetic relationships. So, it can be used in the assignment of close relationships at the genus level [12], and in several cases at the species level [39, 40]. Phylogenetic analysis using maximum likelihood method based on Tamura-Nei model [26] showed that the closest strain to our isolate is P. mirabilis identified in India (EU411047). Sequencing independent techniques such as pulsed- field gel electrophoresis (PFGE) [41, 42], random amplified polymorphism deoxyri- bonucleic acid (RAPD) [43], and restriction fragment length polymorphism (RFLP) [44] have broad availability and cost lower than other typing methods; however, 16S rRNA sequencing has high discriminatory power, 100% typeability and good reproducibility [45]. P. mirabilis isolates were found to be resistant to some antibiotics. A lot of studies detected P. mirabilis as Extended-spectrum beta-lactamases ESBLs producer [46]. A number of studies had focused on antibiotic resistance among bacteria recovered from banknotes [47]. Isolates that are resistant to commonly used antibiotics represents risks and public-health hazards to the community and individuals handling banknotes [48]. 5. Conclusion Our study has shown that some currency notes circulated at Khartoum transportation are carriers of antimicrobial-resistant P. mirabilis that could be potential source for their transmission in public. Awareness about the health risk of contaminated currency and proper hand hygiene might be necessary. Notes sterilization and electronic credit cards use are recommended. Conflict of Interests The authors declare that there is no conflict of interest regarding the publication of this article. 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DOI 10.18502/sjms.v13i3.2955 Page 186 Introduction Materials and Methods Sources of banknotes Phenotypic analysis of bacteria from Sudanese Banknotes Bacterial extraction and biochemical tests Susceptibility tests Genotypic analysis of bacteria from Sudanese Banknotes DNA extraction 16S rRNA gene amplification Detection of amplified product by agarose gel electrophoresis Bioinformatics analysis Results Results of susceptibility tests DNA quantification results Fragments separation using gel electrophoresis Multiple sequence alignment Phylogenetic analysis Discussion Conclusion Conflict of Interests Acknowledgments References