International Journal of Microbiology

International Journal of Microbiology / 2020 / Article

Research Article | Open Access

Volume 2020 |Article ID 1231807 | 5 pages | https://doi.org/10.1155/2020/1231807

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

Academic Editor: Joseph Falkinham
Received26 Sep 2019
Revised06 Dec 2019
Accepted13 Jan 2020
Published07 Feb 2020

Abstract

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 () 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.

1. Introduction

Proteus mirabilis is one of the most common Gram-negative 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]. The 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. This 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]. The 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). The 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]. The rsbA gene expresses a histidine-containing phosphotransmitter of the bacterial two-component signaling system. This 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]. The Mannose-resistant/Proteus-like fimbriae (MR/P) are related to bladder and kidney infection [9]. The mr/p gene cluster comprised two transcripts: mrpABCDEFGHJ (operon) and mrpI. The 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]. The 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]. The 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].

Thus, 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.

2. 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 (>105 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].

2.2. Antimicrobial Susceptibility Testing

The following antimicrobials were used in the current study: ciprofloxacin (5 μg, Hikma Pharmaceutical, Jordan), cephalexin (30 μg, Dar Al Dawa, Jordan), nalidixic acid (30 μg, Hikma Pharmaceutical, Jordan), sulfamethoxazole (25 μg, Dar Al Dawa, Jordan), ceftriaxone (30 μg, Pfizer, USA), nitrofurantoin (300 μg, Jordan River Pharmaceutical Industries, Amman), norfloxacin (10 μg, Amman Pharmaceutical industries, Jordan), ampicillin (10 μg, Jordan Veterinary and Agriculture Medical Industrial Company, Amman), and gentamicin (10 μg, Hikma Pharmaceutical, Jordan).

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 × 108 organisms per milliliter as determined by McFarland standard scale number 0.5 [14]. The cultures were uniformly streaked onto fresh Mueller Hinton agar plates using sterile cotton swabs. The plates were allowed to dry-off briefly, and then the discs of different antimicrobials were mounted onto the surface of the streaked inoculums. The plates were incubated at 37°C for 24 hours. Then, the culture plates were examined for inhibition. The zones of growth inhibition were measured using a meter rule described previously [15].

2.3. 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.

2.4. Molecular Detection of P. mirabilis Virulence Genes

Several virulence genes were detected using conventional PCR amplification. The PCR cycling protocol and primer sequences for each gene were previously described [11, 1720]. Confirmation of gene identity relied on finding a band corresponding to expected PCR product size.

2.5. 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. The Pearson Chi-squared test was used to compare frequency data. value less than 0.05 was considered statistically significant.

3. Results

3.1. Antimicrobial Susceptibility Results

The 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. The rate of antibiotic resistance was highest for nitrofurantoin (88.9%), whereas it was lowest for ceftriaxone (1.6%).


Antimicrobial agentSusceptible (%)Intermediate (%)Resistant (%)

Ciprofloxacin44 (69.8%)11 (17.5%)8 (12.7%)
Cephalexin33 (52.4%)9 (14.3%)21 (33.3%)
Nalidixic acid27 (42.9%)7 (11.1%)29 (46%)
Sulfamethoxazole25 (39.7%)3 (4.8%)35 (55.6%)
Ceftriaxone61 (96.8%)1 (1.6%)1 (1.6%)
Nitrofurantoin2 (3.2%)5 (7.9%)56 (88.9%)
Norfloxacin52 (82.5%)4 (6.3%)7 (11.1%)
Ampicillin23 (36.5%)1 (1.6%)39 (61.9%)
Gentamicin45 (71.4%)5 (7.9%)13 (20.6%)

3.2. P. mirabilis Urovirulence Genes

Detected rates of virulence genes are shown in Table 2. The 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 ().


GenePresent (%)

hpmA62 (98.4%)
hpmB63 (100%)
rsbA63 (100%)
luxS63 (100%)
ureC160 (95.2%)
hlyA15 (23.8%)
rpoA61 (96.8%)
atfA62 (98.4%)
atfC60 (95.2%)
mrpA58 (92.1%)
pm158 (92.1%)


Antibiotics/genesCIPCLNASMXCTXNFTNFXAMPGN

hpmASIRSIRSIRSIRSIRSIRSIRSIRSIR
100001001001001100100001100
+431183392027728253346110155651472313844513
value0.8030.3620.5510.6660.0000.0000.8980.7320.816
hpmB000000000000000000000000000
+441183392127729253356111255652472313945513
value
rsbA000000000000000000000000000
+441183392127729253356111255652472313945513
value
luxS000000000000000000000000000
+441183392127729253356111255652472313945513
value
ureC1120003003102201120300003300
+43983391827726243335910135649472313642513
value0.0680.0430.1580.8820.0000.0000.7170.3790.533
hlyA34952291718624182284701244239451812933411
+1023110491571714100114130250101212
value0.5920.0940.3030.7150.1710.7020.5020.8030.687
rpoA110002002101101110200002200
+431083391927727243346010145650472313743513
value0.4420.1270.2980.9210.0000.0000.8040.5300.662
atfA100001001010100100100001100
+431183392026729252356011155651472313844513
value0.8030.3620.5080.0000.9830.0000.8980.7320.816
atfC120003003102201120300003300
+43983391827726243335910135649472313642513
value0.0680.0430.1580.8820.0000.0000.7170.3790.533
mrpA311113014212401113401104401
+411073281827625232335710145348462213541512
value0.8510.3060.1300.2360.0030.0420.6920.6770.784
pm1221221014203401113401203401
+42973181927625233325710145348462113641512
value0.2860.8370.1300.8700.0030.0420.6920.9480.784

Ciprofloxacin: CIP, cephalexin: CL, nalidixic acid: NA, sulfamethoxazole: SMX, ceftriaxone: CTX, nitrofurantoin: NFT, norfloxacin: NFX, ampicillin: AMP, and gentamicin: GN.

4. 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. The 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.

The 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.

Most isolates were resistant to nitrofurantoin (88.9%), ampicillin (61.9%), and sulfamethoxazole (55.6%). On the contrary, the highest sensitivity was against ceftriaxone (96.8%), norfloxacin (82.5%), gentamicin (71.4%), and ciprofloxacin (69.8%). Similar results have been reported for P. mirabilis from Nigeria, where isolates’ resistance rates to ciprofloxacin, nalidixic acid, sulfamethoxazole, and gentamicin were 13.9%, 53.7%, 74.1%, and 26.9%, respectively [21]. In Czech Republic, the isolates had a resistance rate of ciprofloxacin (35.2%), sulfamethoxazole (39.0%), ampicillin (38.5%), and gentamicin (25.4%), which are different form findings of the present study [22]. The noticed variations in resistance rates may be referred to regional variation in bacterial strain and virulence genes prevalence, in addition to different standards and controls for prescription and use of antimicrobial agents.

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. There was coassociation between hpmA, ureC1, rpoA, atfC, mrpA, and pm1 genes. Therefore, 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. The 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]. The previously mentioned prevalence rates most likely attribute to the differences in the distribution of virulence genes among different populations and geographic locations. The 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

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

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This project was funded by Deanship of Research at Jordan University of Science and Technology.

References

  1. J. N. Schaffer and M. M. Pearson, “Proteus mirabilis and urinary tract infections,” Microbiology Spectrum, vol. 3, 2015. View at: Publisher Site | Google Scholar
  2. R. T. Jamil, L. A. Foris, and J. Snowden, “Proteus mirabilis infections,” StatPearls, Treasure Island, FL, USA, 2019. View at: Google Scholar
  3. M. Umar, A. Arzai, G. Yusuf et al., “Serological characterization and antimicrobial sensitivity profile of Haemophilus influenzae serotypes isolated from aminu kano teaching hospital, kano, Nigeria,” British Microbiology Research Journal, vol. 15, no. 5, pp. 1–10, 2016. View at: Publisher Site | Google Scholar
  4. K. F. Abbas, J. K. Al Khafaji, and M. S. Al-Shukri, “Molecular detection of some virulence genes in Proteus mirabilis isolated from hillaprovince,” International Journal of Research Studies in Biosciences, vol. 3, pp. 85–89, 2015. View at: Google Scholar
  5. R. M. Morgenstein, B. Szostek, and P. N. Rather, “Regulation of gene expression during swarmer cell differentiation in Proteus mirabilis,” FEMS Microbiology Reviews, vol. 34, no. 5, pp. 753–763, 2010. View at: Publisher Site | Google Scholar
  6. C. E. Armbruster, H. L. T. Mobley, and M. M. Pearson, “Pathogenesis of Proteus mirabilis infection,” EcoSal Plus, vol. 8, no. 1, 2018. View at: Publisher Site | Google Scholar
  7. C. E. Armbruster and H. L. T. Mobley, “Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis,” Nature Reviews Microbiology, vol. 10, no. 11, pp. 743–754, 2012. View at: Publisher Site | Google Scholar
  8. J. N. Schaffer, A. N. Norsworthy, T.-T. Sun, and M. M. Pearson, “Proteus mirabilis fimbriae-and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation,” Proceedings of the National Academy of Sciences, vol. 113, no. 16, pp. 4494–4499, 2016. View at: Publisher Site | Google Scholar
  9. R. Pellegrino, U. Galvalisi, P. Scavone, V. Sosa, and P. Zunino, “Evaluation of Proteus mirabilis structural fimbrial proteins as antigens against urinary tract infections,” FEMS Immunology & Medical Microbiology, vol. 36, no. 1-2, pp. 103–110, 2003. View at: Publisher Site | Google Scholar
  10. A. N. Norsworthy and M. M. Pearson, “From catheter to kidney stone: the uropathogenic lifestyle of Proteus mirabilis,” Trends in Microbiology, vol. 25, no. 4, pp. 304–315, 2017. View at: Publisher Site | Google Scholar
  11. S. E. Cestari, M. S. Ludovico, F. H. Martins, S. P. D. da Rocha, W. P. Elias, and J. S. Pelayo, “Molecular detection of HpmA and HlyA hemolysin of uropathogenic Proteus mirabilis,” Current Microbiology, vol. 67, no. 6, pp. 703–707, 2013. View at: Publisher Site | Google Scholar
  12. L. S. Burall, J. M. Harro, X. Li et al., “Proteus mirabilis genes that contribute to pathogenesis of urinary tract infection: identification of 25 signature-tagged mutants attenuated at least 100-fold,” Infection and Immunity, vol. 72, no. 5, pp. 2922–2938, 2004. View at: Publisher Site | Google Scholar
  13. S. L. Chiang and E. J. Rubin, “Construction of a mariner-based transposon for epitope-tagging and genomic targeting,” Gene, vol. 296, no. 1-2, pp. 179–185, 2002. View at: Publisher Site | Google Scholar
  14. S. Irfan, A. Zafar, D. Guhar, T. Ahsan, and R. Hasan, “Metallo-β-lactamase-producing clinical isolates of Acinetobacter species and Pseudomonas aeruginosa from intensive care unit patients of a tertiary care hospital,” Indian Journal of Medical Microbiology, vol. 26, no. 3, pp. 243–245, 2008. View at: Publisher Site | Google Scholar
  15. M. Umar, D. Akafyi, Y. Jobbi, A. Ayaya, and I. Abdulkarim, “Biochemical characterization and antibiogram pattern of Streptococcus mutans isolated from Dental Unit, Sick-Bay, Ahmadu Bello University, Zaria, Nigeria,” International Journal of Biological and Biomedical Sciences, vol. 4, pp. 63–66, 2015. View at: Google Scholar
  16. S. Aghamiri, N. Amirmozafari, J. Fallah, B. Fouladtan, and H. Kafil, “Antibiotic resistance pattern and evaluation of metallo-beta lactamase genes including bla-IMP and bla-VIM types in Pseudomonas aeruginosa isolated from patients in Tehran Hospitals,” ISRN Microbiology, Article ID 941507, 6 pages, 2014. View at: Publisher Site | Google Scholar
  17. S. A. Badi, J. Norouzy, and A. A. Sepahi, “Detection Rsba gene’s band & effect of miristic acid in virulence of Proteus mirabilis isolated from urinary tract infrction,” Iranian Journal of Public Health, vol. 43, p. 210, 2014. View at: Google Scholar
  18. H. S. Huang, J. Chen, L. J. Teng, and M. K. Lai, “Use of polymerase chain reaction to detect Proteus mirabilis and Ureaplasma urealyticum in urinary calculi,” Journal of the Formosan Medical Association, vol. 98, no. 98, pp. 844–850, 1999. View at: Google Scholar
  19. V. Sosa, G. Schlapp, and P. Zunino, “Proteus mirabilis isolates of different origins do not show correlation with virulence attributes and can colonize the urinary tract of mice,” Microbiology, vol. 152, no. 7, pp. 2149–2157, 2006. View at: Publisher Site | Google Scholar
  20. P. Zunino, L. Geymonat, A. G. Allen, C. Legnani-Fajardo, and D. J. Maskell, “Virulence of a Proteus mirabilis ATF isogenic mutant is not impaired in a mouse model of ascending urinary tract infection,” FEMS Immunology & Medical Microbiology, vol. 29, no. 2, pp. 137–143, 2000. View at: Publisher Site | Google Scholar
  21. O. S. Alabi, N. Mendonça, O. E. Adeleke, and G. J. da Silva, “Molecular screening of antibiotic-resistant determinants among multidrug-resistant clinical isolates of Proteus mirabilis from SouthWest Nigeria,” African Health Sciences, vol. 17, no. 2, pp. 356–365, 2017. View at: Publisher Site | Google Scholar
  22. L. Cernohorska and E. Chvilova, “Proteus mirabilis isolated from urine, resistance to antibiotics and biofilm formation,” Klinicka Mikrobiologie a Infekcni Lekarstvi, vol. 17, pp. 81–85, 2011. View at: Google Scholar
  23. P. Scavone, V. Iribarnegaray, A. L. Caetano, G. Schlapp, S. Hartel, and P. Zunino, “Fimbriae have distinguishable roles in Proteus mirabilis biofilm formation,” Pathogens and Disease, vol. 74, 2016. View at: Publisher Site | Google Scholar

Copyright © 2020 Emad I. Hussein et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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