1. Introduction
Proteus mirabilis belongs to the family Enterobacteriaceae and is capable of transforming in shape from rod to elongated and swarming motility by flagella. It is an opportunity bacteria and can cause different clinical diseases, including urinary tract infection, wound infection, meningitis in infants, rheumatoid, endocarditis, septicemia, and cystic fibrosis. This broad infection caused by Proteus mirabilis is produced due to the fact that it contains many virulence factors, including adhesion, toxins, flagella, enzyme production like urase, biofilm and highly resistant phenotype to antibiotics. The regulation of these virulence factor gene expressions is coordinated by signals in a phenomenon called quorum sensing ( 1 - 3 ).
In urinary tract infections, biofilm production leads to long-period infection ( 4 - 6 ).
The Proteus mirabilis genome contains different genes that encode proteins responsible for antibiotic resistance and develop multidrug and extensive resistance strains ( 7 ). These genes may be mobile genes located on chromosomes or plasmids called integrons (IntI1, IntI2) as well as other genes as included in this study that leads to transfer antibiotic resistance horizontally among strains of these bacteria and able Proteus mirabilis to resist different groups of antibiotic (beta-lactam, quinolones, chloramphenicol, trimethoprim, aminoglycoside, rifampicin) in nosocomial infection that attract the attention of world health organization and put this multidrug resistance pathogen in a group of microorganisms that medically significant nosocomial and community-acquired pathogen ( 8 , 9 ). So, this study was carried out to assay and detect a sequence of genes that encode for antibiotic resistance included (blaTEM, aac(6’)-Ib, IntI2, IntI1), which considers resistance genes and include a mobile genetic element in multidrug resistance P. mirabilis in clinical samples and study of the relationship in the phylogenetic tree among these genes that responsible for the resistance of antibiotics.
2. Materials and Methods
2.1. Isolation and Identification of Proteus mirabilis
The study collected 500 clinical infections from urine, wound, burn and otitis. All these samples were distributed among six governorates in Iraq, including Baghdad, Babylon, Al-Anbar, Al- Muthanna, Wasit, and Diyala, from October 2020 to January 2021. Identification of these samples depended on morphological and biochemical tests through appearance swarming, non-lactose fermentation on selective and differentialMacConkey and enriched condition on blood agar to another step that included confirmation of Proteus mirabilis diagnosis by VITEK-2 Compact system (Biomerieuxm France). The confirmed isolates of Proteus mirabilis were 95 clinical isolates.
2.2. Antibiotic Susceptibility Test
All Proteus mirabilis isolates were tested by using"Amoxicillin 10µg/ml, Aztreonam 30µg/ml, Imipenem 10µg/ml, Cefoxitin 30µg/ml, Amikacin 30µg/ml, Ceftazidem 30µg/ml, Ciprofloxacin 5µg/ml, Nalidixic acid 30µg/ml, Gentamicin 10µg/ml, Sulphamethazol-trimethoprim 25µg/ml, Cefotaxim 30µg/ml, Amoxicillin-clavulanic acid 20/10 µg/ml". Then isolates were considered resistant or sensitive depending on the previously published regulation ( 10 ).
2.3. Molecular Study of Target Genes
2.3.1. Multidrug Resistance Proteus mirabilis
Ten more resistant isolates of Proteus mirabilis PM1, PM2, PM3, PM4, PM5, PM6, PM7, PM8, PM9, and PM10 were selected and used for genomic DNA extraction according to manufacturer protocol (Geneaid Extraction Promega, USA). The extracted DNA was concentrated at 27 ng/µl.
2.3.2. Primers Used in This Study
Specific primers for the detection of genes responsible for the resistance of antibiotics, as shown below in table 1, were used.
| Primer Name | Seq. | Annealing Temp. (°C) | Product Size (bp) |
|---|---|---|---|
| aacIb-F | 5`-TTGCGATGCTCTATGAGTGGCTA-3` | 54 | 482 (11) |
| aacIb-R | 5`-CTCGAATGCCTGGCGTGTTT-3 | ||
| blaTEM-F | 5`-TACGATACGGGAGGGCTTAC-3 | 52 | 716 (12) |
| blaTEM-R | 5`-TTCCTGTTTTTGCTCACCCA-3 | ||
| IntI1-F | 5`-CAGTGGACATAAGCCTGTTC-3 | 59 | 160 (13) |
| IntI1-R | 5`-CCCGAGGCATAGACTGTA-3 | ||
| IntI2-F | 5`-CACGCATATGCGACAAAAAGGT-3 | 55 | 788 (13) |
| IntI2-R | 5`-GTAGCAAACGAGTGACGAAATG-3 |
2.4. Sequencing of Resistance Genes
PCR products of resistance genes (aacIb, qnrA, blaTEM, IntI1, IntI2) were electrophoresis on gel against 100 bp marker of DNA ladder. Then, nucleotide sequencing of these genes and selecting some important sequences of genes are registered in NCBI.
3. Results
3.1. Identification of Proteus mirabilis in Clinical Samples
Standard methods and biochemical tests tested all 500 clinical samples, and then identification was confirmed by VITEK-2 Compact System. The results showed that 95 isolates (19%) belong to Proteus mirabilis from different regions in Iraq (Table 2).
| Type of Clinical Sample | Number of isolates | Percentage |
|---|---|---|
| Infection of burn | 21 | 22.1% |
| ear swabs | 22 | 23.25% |
| Midstream urine | 52 | 54.7% |
| Total | 95 | 100% |
3.2. Antibiotic Resistance Pattern
Ninety-five Proteus mirabilis isolates were examined to detect multidrug and extensive drug-resistant against twelve antibiotics, including amoxicillin, Aztreonam, Imipenem, Cefoxitin, Amikacin, Ceftazidem, Ciprofloxacin, Nalidixic acid, Gentamicin, Sulphamethazol-trimethoprim, Cefotaxim, and Amoxicillin-clavulanic acid. The results of antibiotics resistance exhibited the ability of Proteus mirabilis to resist antibiotics at different levels, as shown in table 3, then 10 more resistant isolates were selected (Table 4) for molecular study. The results of the antibiotic resistance pattern are shown in table 3.
| Antimicrobial Agent | Proteus mirabilis (95 isolates) | |||
|---|---|---|---|---|
| Resistance | Sensitive | |||
| NO. | % | NO. | % | |
| Amoxicillin (AMX) 10µg/ml | 47 | 49.47% | 48 | 50.5% |
| Aztreonam (ATM) 30µg/ml | 6 | 6.3% | 89 | 93.68) |
| Imipenem (IPM) 10µg/ml | 0 | 0% | 95 | 100% |
| Cefoxitin (FOX) 30µg/ml | 17 | 17.89% | 78 | 82.1% |
| Amikacin (AK)30µg/ml | 10 | 10.5% | 85 | 89.47% |
| Ceftazidem (CAZ) 30µg/ml | 29 | 30.5% | 66 | 69.47% |
| Ciprofloxain (CIP) 5µg/ml | 8 | 8.4% | 87 | 91.57% |
| Nalidixic acid( NA) 30µg/ml | 42 | 44.2% | 52 | 54.73% |
| Gentamicin (CN) 10µg/ml | 24 | 25.26% | 71 | 74.73% |
| Sulphamethazol-trimethoprim (SXT) 25µg/ml | 64 | 67.36% | 31 | 32.63% |
| Cefotaxim (CTX) 30µg/ml | 42 | 44.2% | 53 | 55.78% |
| Amoxicillin-clavulanic acid (AUG) 20/10 µg/ml | 45 | 47.36 | 50 | 52.63 |
| No. of isolate | Antimicrobial Agent | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AMX 10µg/ml | ATM 30µg/ml | IPM 10µg/ml | FOX 30µg/ml | AK 30µg/ml | CAZ 30µg/ml | CIP 5µg/ml | NA 30µg/ml | CN 10µg/ml | SXT 25µg/ml | CTX 30µg/ml | AUG 20/10 µg/ml | |
| PM 1 | R | R | S | R | S | R | S | R | R | R | R | R |
| PM 2 | R | R | S | R | R | R | R | R | S | R | R | R |
| PM 3 | R | S | S | S | S | R | S | R | R | R | R | R |
| PM 4 | R | R | S | R | R | R | R | R | R | R | R | R |
| PM 5 | R | S | S | S | S | R | R | R | S | R | R | R |
| PM 6 | R | R | S | R | S | R | S | R | R | R | R | R |
| PM 7 | R | S | S | R | R | R | S | R | R | R | R | R |
| PM 8 | R | R | S | R | R | R | R | R | S | S | R | R |
| PM 9 | R | S | S | S | S | R | R | R | R | R | R | R |
| PM 10 | R | S | S | S | S | R | R | R | S | R | R | R |
| R = resistant S = Sensitive | ||||||||||||
3.3. DNA Extraction from Multidrug Resistance Proteus mirabilis
The 10 multidrug-resistant isolates of Proteus mirabilis tabulated in table 4 were used for more molecular investigation. The results showed all ten multidrug resistance Proteus mirabilis contain these genes in different levels blaTEM gene (90%), aac(6’)-Ib gene (80%), IntI1 gene (100%), IntI2 gene (80%) as shown in figures (1, 2, 3 and 4).
Figure 1.aac(6)-lb (482bp) resistant P. mirabilis comparison with stander marker (1500 bp)
Figure 2.blaTEM (716 bp) in resistant P. mirabilis comparison with stander marker (1500 bp)
Figure 3.IntI-1(160 bp) in resistant P. mirabilis comparison with stander marker (1500 bp)
Figure 4.IntI-2 (788bp) in resistant P. mirabilis comparison with stander marker (1500 bp)
3.4. Sequence of MDR P. mirabilis genes
The sequencing of multidrug resistance Proteus mirabilis (32 isolates) containing genes including aacIb, blaTEM, IntI1, and IntI2 were sent for Sanger sequencing to Macrogen Corporation – Korea. Geneious software analyzed the results to detect a mismatch with P. mirabilis in NCBI, as shown in figures 5, 6, 7 and 8.
Figure 5. Dendrogram of sequence aacIb in P. mirabilis (4, 6, 7, 8) comparison with world Proteus mirabilis in NCBI (CP061834, CP061843, CP062793)
Figure 6. Dendrogram of sequence TE Min P. mirabilis (2, 3, 4, 5, 7, 8, 9, 10) comparison with world Proteus mirabilis in NCBI (MK861851, MN167852)
Figure 7. Dendrogram of sequence IntI1 in P. mirabilis (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) comparison with world Proteus mirabilis in NCBI (MN167852, MK847916, MK847915)
Figure 8. Dendrogram of sequence IntI2 in P. mirabilis (2, 4, 5, 6, 7, 8, 9, 10) comparison with world Proteus mirabilis in NCBI (MK670987, CP059056, CP063314)
Documentation of genes from Iraqi multidrug-resistant clinical isolates of Proteus mirabilis carried out as new Iraqi strains. They selected five isolates of multidrug resistance Proteus mirabilis with specific sequences of nucleotides with genetic variation compared with world strain. The results showed an accepted sequence of five genes for five isolates in NCBI, including the following genes aacIb (LC613168.1), blaTEM (LC613166.1), IntI1 (LC613169.1), IntI2 (LC613170.1). All mentioned strains in this research are named (Iraq 2-4-5-6).
4. Discussion
Proteus mirabilis became a prevalent opportunistic pathogen agent in clinical infection worldwide and is associated with UTI especially among patients with long-term indwelling catheters, burn infections, ear infections, and septicemia ( 14 , 15 ). In Iraqi clinical isolates, infections with Proteus mirabilis were burned infection 21 (22.1%), ear infection 22 (23.25%), and urinary tract infection 52 (54.7%). The spreading of infection with these bacteria belongs to many virulence factors and a high ability to resist different antibiotics ( 16 ).
Multidrug resistance ten Proteus mirabilis isolates have different levels of all resistance genes (blaTEM, aac(6’)-Ib, IntI2, IntI1). Genome of nine isolates of these bacteria contain blaTEM gene (90%), eight isolates contain aac(6’)-Ib gene (80%) ,all isolates contain IntI1 gene (100%), eight isolates contain IntI2 gene (80%). These above genes make Proteus mirabilis isolates able to resist different groups of antibiotics likeβ-lactam, aminoglycoside, and quinolones, as shown in table 4. Analysis sequencing of the above target genes may be referred to as these genes' horizontal transport among Proteus mirabilis subspecies because it is located in some groups in a phylogenetic tree.
Also, the phylogenetic tree exhibited sequences of genes variable comparison with global strain in NCBI of the same bacteria locally. The selected five Iraqi strains were to interpret the origin of this genetic variation that may occur due to mutation or integrons ( 17 , 18 ) and accepted as Iraqi strains in NCBIincludedaacIb (LC613168.1), blaTEM (LC613166.1), IntI1 (LC613169.1), IntI2 (LC613170.1).
Proteus mirabilis produces extended-spectrum β-lactamase enzyme developed as a result of a mutation that occurs in β-lactamase like (TEM-1, CTX-M, SHV-1), and this mutation may be point mutation leading to a change in type and sequence amino acid in protein peptide. These enzymes consider primary causal agents that lead to increased resistance against the β-lactam group, especially cephalosporins antibiotics that include (cefotaxime, ceftazidime), and aztreonam ( 19 , 20 ). All that spreading of multidrug resistance in these bacteria in the clinical environment is due to the random use of antibiotics ( 21 , 22 ).
The ability of Proteus mirabilis to resist quinolones may occur because chromosomal mutation affects gyr A, topoisomerase genes and plasmid genes like qnr genes ( 23 , 24 ). These genes encode proteins that act on the target site to quinolones and cause the inhibition effect of this group of antibiotics, especially norfloxacin and ciprofloxacin ( 11 , 25 - 27 ).
The aminoglycoside group is significant in treatment because it includes a broad spectrum of antibiotics ( 28 , 29 ). The isolates Proteus mirabilis contain aac(6’)-Ib gene that encodes to an aminoglycoside-modifying enzyme that acts inhibition effect against antibiotics in this group like amikacin and gentamycin, then isolates of these pathogenic bacteria become resistant against antibiotic of aminoglycoside group ( 30 - 32 ).
The genome of Proteus mirabilis contains mobile genetic elements like integrons, including IntI1 and IntI1 genes. These integrons contain three regions, a gene encoding to production integrase enzyme, the primary site for the recombination stage, and a promotor regulating the transcription process of captured genes that encode proteins, making these bacterial isolates able to resist antibiotics ( 33 - 35 ).
Analysis of the phylogenetic tree showed genetic variation between Iraqi clinical Proteus mirabilis and global, documented in NCBI. Genetic variation in the sequence of aacIb gene from Iraq – 4 infection UTI documented clinically data NCBI (LC613168.1). Sequencing of the blaTEM gene refers to found genetic variation in Iraq 2 strain that was isolated from midstream urine and accepted in NCBI (LC613166.1). The nucleotides of the IntI1 gene detected in Iraq 5 strain isolated from burn infection and registered in NCBI (LC613169.1). The difference between Iraqi and global sequencing in IntI2 was achieved in Iraq 6strain, isolated from midstream urine, and then documented in NCBI (LC613170.1). Found a high degree of similarity in the sequence of antibiotic resistance genes (blaTEM, aac(6’)-Ib, IntI2, IntI1) from locally clinical isolates Proteus mirabilis may be referred to horizontal transport of these genes among species of these pathogenic bacteria.
Authors' Contribution
Study concept and design: A. S. H.
Acquisition of data: E. A. G.
Analysis and interpretation of data: A. S. H.
Drafting of the manuscript: E. A. G.
Critical revision of the manuscript for important intellectual content: W. F. H.
Statistical analysis: A. S. H.
Administrative, technical, and material support: A. S. H.
Conflict of Interest
The authors declare that they have no conflict of interest.
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