Proteus mirabilis causes 90% of Proteus infections . It has been implicated in meningitis, empyema, osteomyelitis, and gastroenteritis. Also, it frequently causes health care-associated infections of the urinary tract, surgical wounds and lower respiratory tract . Identification of bacteria is frequently performed by isolation of the organisms and study of their phenotypic characteristics, including gram staining, morphology, culture requirements, and biochemical reactions . 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 making the identification of fastidious and unculturable bacteria difficult and sometimes impossible . Moreover, phenotypic systems cannot account for the variable characteristics observed among members of the same species, resulting in poor precision upon repeated testing . Construction of phylogeny tree based on phenotypic methods 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 . In addition to the fact that phenotype represents a very small part of each organism's genome .
Specific properties of the 16S rRNA gene include its ubiquitous distribution, mosaic structure , and relative stability that qualify it to be used in the taxonomic assignment and phylogenetic relationship determination . The genotypic bacterial identification 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 . Direct sequencing of 16S rRNA genes from environmental samples has become a standard and convenient method of assessing microbial population abundance , structure, and function in microbial communities [16,17].
This study aimed to identify the bacterial isolates recovered from Sudanese banknotes using culture-based and 16S rRNA gene sequencing techniques.
2. Materials and Methods
2.1. Sources of banknotes
Prospective study was conducted in March 2016 in which a total of 45 Sudanese banknotes 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 aerobically 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 .
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, Ceftriaxone (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, Chloramphenicol (C) 30 µg, and Meropenem (MEM) 10 µg using Kirby–Bauer disc diffusion method .
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  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')  were used to amplify 16S rRNA gene. The PCR reaction 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 . 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  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  and subjected for multiple sequence alignment using Clustal W program  within BioEdit software , with total alignment score 1661794. The phylogenetic tree was constructed according to maximum likelihood method using MEGA 7 software .
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.
Table 1 shows the results of susceptibility test of P. mirabilis isolates toward commonly used antibiotics using disc diffusion assay.
3.2. DNA quantification results
|Sample No.||Protein µg/l||Ratio||ssDNA µg/l||Purity %|
Table 2 illustrates the DNA concentrations of five samples using GeneQuant machine. Only Sample 5 was adopted for further processes.
3.3. Fragments separation using gel electrophoresis
Figure 1 represents separated band of Sample 5 compared with bands of ladder using gel electrophoresis.
3.4. Multiple sequence alignment
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 . 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.
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 frequent exchange, poor hygiene and environmental conditions such as temperature and humidity . 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 phenotypic traits . 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 . After quantification of extracted DNA, gel electrophoresis and DNA sequencing, the obtained 756 pb sequence with the aid of BLAST and multiple sequence alignment 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 . 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 , and in several cases at the species level [39,40]. Phylogenetic analysis using maximum likelihood method based on Tamura-Nei model  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 deoxyribonucleic acid (RAPD) , and restriction fragment length polymorphism (RFLP)  have broad availability and cost lower than other typing methods; however, 16S rRNA sequencing has high discriminatory power, 100% typeability and good reproducibility .
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 . A number of studies had focused on antibiotic resistance among bacteria recovered from banknotes . Isolates that are resistant to commonly used antibiotics represents risks and public-health hazards to the community and individuals handling banknotes .
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.