Assessment of Pathogenic Potential, Virulent Genes Profile, and Antibiotic Susceptibility of Proteus mirabilis from Urinary Tract Infection

Proteus mirabilis is the third most common bacterium that can cause complicated UTI, especially in catheterized patients. Urovirulence genes of P. mirabilis strains are poorly identified among UTI patients. The aims of the present study were to determine the prevalence of the uropathogenic P. mirabilis strains isolated from UTI patients by the detection of several P. mirabilis virulence genes and to characterize the antibiotic susceptibility profile of P. mirabilis isolates. P. mirabilis isolates were collected from urine specimens of patients suffering from UTI. Virulence genes in P. mirabilis, namely, hpmA, hpmB, rsbA, luxS, ureC1, hlyA, rpoA, atfA, atfC, mrpA, and pm1 were detected in the isolates via PCR detection method. All P. mirabilis virulence genes were detected in more than 90% of the isolates except hlyA gene, which was detected in only 23.8% of the isolates. The rate of susceptibility for ceftriaxone was 96.8%, followed by norfloxacin (82.5%), gentamicin (71.4%), ciprofloxacin (69.8%), cephalexin (52.4%), nalidixic acid (42.9%), sulfamethoxazole (39.7%), ampicillin (36.5%), and nitrofurantoin (3.2%). Significant associations (P < 0.05) were detected between antimicrobial susceptibility of each of the following antibiotics and the presence virulence genes. Cephalexin antimicrobial susceptibility was significantly associated with the presence each of ureC1 and atfC. Sulfamethoxazole antimicrobial susceptibility was significantly associated with the presence atfA. Ceftriaxone antimicrobial susceptibility was significantly associated with the presence each of hpmA, ureC1, rpoA, atfC, mrpA, and pm1. Nitrofurantoin antimicrobial susceptibility was significantly associated with the presence each of hpmA, ureC1, rpoA, atfA, atfC, mrpA, and pm1. In conclusion, an association between the presence of urovirulence genes of P. mirabilis and increasing P. mirabilis resistance to antimicrobials has been demonstrated.


Introduction
Proteus mirabilis is one of the most common Gramnegative bacteria that can cause UTIs. Bacteriuria, kidney stones, catheter obstruction, acute pyelonephritis, and fever can be developed by P. mirabilis [1]. In fact, P. mirabilis strains are responsible for the majority of complicated urinary tract infections [1]. P. mirabilis is becoming resistant to antibiotics commonly used in the treatment of UTI [2]. e bacterium was shown to be highly sensitive to streptomycin (100%), erythromycin (85%), and sparfloxacin (75%), whereas it showed high resistance to amoxicillin (100%), tetracycline (95%), and cefuroxime (80%). Isolated Proteus mirabilis has shown multiple drug-resistance ability to the used antibiotics [3].
P. mirabilis encodes many virulence genes involved in infection [4,5]. Urease (ureC1) is a virulence gene that is important in P. mirabilis pathogenesis. is enzyme catalyzes the kidney and bladder stone formation or blocks indwelling urinary catheters [6]. Urease is required for urolithiasis, where it contributes in hydrolyzing urea to release ammonia, thereby increasing urinary pH, resulting in precipitation of calcium and magnesium compounds, and urinary stone formation [7]. e alteration of pH is important in catheter colonization of P. mirabilis; facilitating the bacterial adherence and biofilm formation [4,8]. Another group of virulence genes is the quorum sensing (luxS, and rsbA). e luxS gene produces signal that is used to sense the interaction of species and its cell density in a polymicrobial community that plays critical roles in the virulence genes regulation [5]. e rsbA gene expresses a histidine-containing phosphotransmitter of the bacterial two-component signaling system.
is gene regulates the swarming manner, which encodes a sensory and act as a protein sensor of environmental circumstances [5]. Subsequently, rsbA facilitated biofilm and extracellular polysaccharide formation [4].
e Mannose-resistant/Proteus-like fimbriae (MR/P) are related to bladder and kidney infection [9]. e mr/p gene cluster comprised two transcripts: mrpABCDEFGHJ (operon) and mrpI. e main structural subunit is mrpA protein, that is required at the first step of infection, including formation of clusters, and is important for wild-type levels of bladder colonization at the following steps [10]. e hemolytic activity of P. mirabilis is related to hemolysin hpmA and hpmB proteins. hpmA is mainly responsible for tissue damage, and hpmA becomes active after cleavage of its N-terminal peptide [6]. e activation and transportation of hpmA depend on hpmB hemolysin [6]. Previous studies suggest that hemolysin plays a critical role in UTI caused by P. mirabilis, which contributes to the potential urovirulence of P. mirabilis [11]. Another type of hemolysin proteins that Proteus species can encode and express is hlyA, and Proteus can encode hlyA gene similar to that virulence gene of E. coli [11].
us, there are many virulence genes that assist survival of P. mirabilis within the urinary system such as urease, hemolysin, fimbriae, and flagella [12]. However, P. mirabilis strains differ in the range and expression levels of virulence genes that can affect growth of bacteria and persistence within the urinary tract. A number of studies have investigated the virulence characteristics of P. mirabilis and mechanisms involved in pathogenesis of UTI to identify the range of P. mirabilis virulence genes and their prevalence among P. mirabilis isolates [4]. In the present study, P. mirabilis isolates involving in human UTI were characterized to identify virulence gene markers in an effort to explore strategies involved in P. mirabilis pathogenesis and antibiotics susceptibility.

Materials and Methods
2.1. P. mirabilis Isolates. P. mirabilis isolates were collected from urine samples of patients who had UTIs and significant bacterial counts (>10 5 CFUs/mL) as per institutional ethics committee approval. Pure cultures were stored at − 80°C in Luria Bertani (LB) broth with 10% glycerol [13]. Samples were collected from July to December 2017 from Jordanian Royal Medical Services. P. mirabilis was identified as per standard diagnostic criteria using its known characteristic of swarming motility and inability to metabolize lactose on a MacConkey agar plate [8].

Antimicrobial Susceptibility Testing.
e following antimicrobials were used in the current study: ciprofloxacin Kirby-Bauer disk diffusion method was used to determine the susceptibility of bacteria to antibiotic agents. Bacterial colonies were transferred from the nutrient agar plate into bottles containing NaCl 0.9% to obtain bacterial density of 1.5 × 10 8 organisms per milliliter as determined by McFarland standard scale number 0.5 [14]. e cultures were uniformly streaked onto fresh Mueller Hinton agar plates using sterile cotton swabs. e plates were allowed to dry-off briefly, and then the discs of different antimicrobials were mounted onto the surface of the streaked inoculums. e plates were incubated at 37°C for 24 hours. en, the culture plates were examined for inhibition. e zones of growth inhibition were measured using a meter rule described previously [15].

Extraction of Genomic DNA.
For all isolates, several bacterial colonies were inoculated in 5 mL Luria Bertani (LB) broth media followed by incubation for 18 hours at 37°C. 1.5 mL of overnight Luria broth bacterial growth culture was subjected to DNA extraction using genomic DNA isolation kit OMEGA bacterial DNA purification kit [16]. Isolated DNA samples were stored at − 20°C till later use.

Molecular Detection of P. mirabilis Virulence Genes.
Several virulence genes were detected using conventional PCR amplification. e PCR cycling protocol and primer sequences for each gene were previously described [11,[17][18][19][20]. Confirmation of gene identity relied on finding a band corresponding to expected PCR product size.

Statistical Analysis.
For the present study, statistical analysis of data using appropriate programs and methods such as the Statistical Package for the Social Sciences (SPSS) version 23 was performed to generate descriptive analysis of raw data, including generation of all frequency tables and cross tabulations. e Pearson Chi-squared test was used to compare frequency data. P value less than 0.05 was considered statistically significant.

Antimicrobial Susceptibility Results.
e antimicrobial susceptibility results of the P. mirabilis isolates to several antimicrobial agents are shown in Table 1, which represents results as susceptible, intermediate, and resistant. e rate of antibiotic resistance was highest for nitrofurantoin (88.9%), whereas it was lowest for ceftriaxone (1.6%).

P. mirabilis Urovirulence Genes.
Detected rates of virulence genes are shown in Table 2. e antimicrobial susceptibility was highly correlated with the presence of P. mirabilis virulence genes (Table 3). Moreover, hpmA, ureC1, rpoA, atfC, mrpA, and pm1 urovirulence genes were more likely to coexist with each other at P. mirabilis (P < 0.001).

Discussion
In the current study, P. mirabilis isolated from UTI patients were analyzed for the presence of virulence genes and susceptibility to antimicrobials. P. mirabilis genes associated with UTIs may be valuable in developing strategies for treating and preventing UTIs. e results of this study provide evidence supporting the role of urovirulence genes of P. mirabilis in human UTIs.
Current results showed the association between resistance to certain antibiotics and the presence of P. mirabilis urovirulence genes. For example, ureC1 and atfC genes were associated with resistance to cephalexin, atfA with resistance to sulfamethoxazole, hpmA, ureC1, rpoA, atfC, mrpA, and pm1 with resistance to ceftriaxone, and hpmA, ureC1, rpoA, atfA, atfC, mrpA, and pm1 with resistance to nitrofurantoin. e correlation between presence of these genes and the increase in resistance toward antibiotics may be attributed to pathogenicity of these genes and their functional roles such as urease, hemolysins, and fimbriae that help the organism to overcome host defense mechanisms and colonize the urinary tract. Overall, these results explain the potential of these uropathogens to interfere with the infection treatment, impair the action of host immune cells, and weaken the antibiotic efficiency.
Urovirulence genes of P. mirabilis strains are poorly identified among UTI patients. One of the aims of this study was to identify the urovirulence genes of P. mirabilis strains isolated from UTI symptomatic patients. Specifically, we investigated the presence of urovirulence genes hpmA, hpmB, rsbA, luxS, ureC1, hlyA, rpoA, atfA, atfC, mrpA, and pm1 using PCR-based analysis. Certain patterns of virulence genes and distributions were identified among the isolates. Statistically significant associations were observed among the P. mirabilis urovirulence genes, as some genes were more likely to coexist with other genes. ere was coassociation between hpmA, ureC1, rpoA, atfC, mrpA, and pm1 genes. erefore, it is likely that a frequent occurrence of antimicrobial resistance is due to the presence of multiple resistance genes that increase the P. mirabilis pathogenicity.
Virulence genes were detected at the following rates among the isolates: hpmB, rsbA, and luxS at 100%, hpmA and atfA at 98.4%, rpoA at 96.8%, ureC1 and atfC at 95.2%, mrpA and pm1 at 92.1%, and hlyA at 23.8%. Some of these prevalence rates are different from those reported from other countries [4,11,22]. Prevalence of these genes may vary according to the clinical status of the host and the genetic makeup of the isolates causing UTIs. e hpmB, luxS, and rsbA genes were the most prevalent at 100%, followed by hpmA and atfA at 98.4% each, while the hlyA gene was the least prevalent at 23.8%. Other urovirulence genes were prevalent in 92-97% of the isolates. Additionally, the high prevalence of hpmB and hpmA at 100% and 98.4%, respectively, in the present study was  consistent with a previous report from Brazil [11]. On the contrary, the prevalence of hlyA (23.8%) is different from the same study, which confirmed that none of the isolates presented hlyA gene [11]. Interestingly, another study from Iraq reported ureC1, mrpA, pm1, luxS, and rsbA prevalence rates of 18%, 35%, 41%, 47%, and 53%, respectively, which are not comparable to our findings [4], whereas a study from Iran reported luxS, and rsbA prevalence rate of 70% each [17]. e previously mentioned prevalence rates most likely attribute to the differences in the distribution of virulence genes among different populations and geographic locations. e current study has some limitations including that it tested only certain virulence genes and certain antibiotics. Studying more virulence genes and antibiotics is recommended future study.
None of the previous studies have investigated a role of atfA and atfC urovirulence genes in P. mirabilis UTIs. Current results suggest that ATF fimbriae could have an important role in adhesion and biofilm formation on abiotic [20,23].
In conclusion, P. mirabilis isolates demonstrated high susceptibility against ceftriaxone, norfloxacin, gentamicin, and ciprofloxacin, and high resistance against nitrofurantoin, ampicillin, and sulfamethoxazole. In addition, significant associations between virulence genes and resistance phenotypes were identified, which suggests increased resistance to antimicrobial agents due to the presence of these virulence genes.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.