year 16, Issue 6 (November - December 2022)                   Iran J Med Microbiol 2022, 16(6): 581-586 | Back to browse issues page


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Shokoohizadeh L, Rabiei M, Baharifar A, Keramat F, Ali L, Alikhani M Y. Evaluation of the Virulence Genes in Quinolone and Fluoroquinolones- resistant Uropathogenic Escherichia coli Isolates. Iran J Med Microbiol 2022; 16 (6) :581-586
URL: http://ijmm.ir/article-1-1699-en.html
1- Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
2- Department of Pharmacology, Hamadan University of Medical Sciences, Hamadan, Iran
3- Brucellosis Research Center, Hamadan University of Medical Sciences, Hamadan, Iran
4- Department of Biological Sciences, National University of Medical Sciences (NUMS), Rawalpindi, Pakistan
5- Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran , alikhani43@yahoo.com
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Introduction


Urinary tract infection (UTI) is one of the most common bacterial infections in the clinical setting, and Uropathogenic Escherichia coli (UPEC) is the major causative pathogen (1). First-line antibiotics for UTI treatment include quinolones (Qs) and fluoroquinolones (FQs) (2). Recent studies showed that the frequency of isolation of UPEC strains resistant to FQs (FQ-R) has increased in recent years (3). UPEC strains can produce various types of virulence factors (4). Studies also suggest that Q-R & FQ-R UPEC strains show reduced virulence and are less capable of causing urinary tract infections than susceptible strains (5). The Qs & FQs have been used to treat a broad spectrum of infections, such as UTIs. A significant increase in QS & FQs- resistant E. coli isolates have been reported (1). The mechanisms of resistance to Q and FQs in E. coli include alterations (mutations) in genes that encoded subunits of DNA gyrase (gyrA & gyrB genes), and topoisomerase IV (parC and parE genes) as the Qs and FQs targets, efflux pump systems and acquisition of plasmid–carrying resistance genes (qnrA, qnrB, qnrS) (6, 7). It has been shown that Q-R, and FQ-R UPEC strains developed a reduced prevalence of virulence factors (VFs) and pathogenesis of UTIs than susceptible strains (8). E. coli expressed three of the major VFS: aerobactin (aer), hemolysin (hly), and P fimbriae (pap). P fimbriae are the second common virulence factor for some pathogenic strains and are encoded by the papA-K operons, which are carried on pathogenicity islands (PAIS). These fimbriae recognize and bind to Galα (1-4) Gal receptors on renal epithelial cells with papG adhesion (9). The most critical secreted VFS for UPEC is α – hemolysin (hlyA), which may cause tissue damage and facilitate bacterial dissemination and release of host nutrients. Hemolysin assists in acquiring iron to regulate the expression of virulence factors (10). Aerobactin is a virulence factor of UPEC strains and other pathogen organisms. Iron acquisition systems utilize siderophores to attract iron for growth and colonize in host iron-poor niches (11). Considering the lack of proper information about the prevalence of virulence factors and fluoroquinolones resistance pattern of UPEC strains in Hamadan city of Iran, these subjects were the main aims of the current research.

 

Materials and Methods

E. coli Identification

One hundred- seventy urine samples were collected consecutively from patients aged 12 to 89 years with an average age of 47 (42 males & 128 females) admitted to three hospitals in Hamadan city, west of Iran. Among 170 urine samples, 125 (73.5%) E. coli strains were isolated. The E. coli isolates were identified by biochemical tests (12) and confirmed by the rpoB gene using PCR (13).

Antimicrobial Susceptibility Testing

The Kirby-Bauer disk diffusion method detected the susceptibility of the UPEC isolates to antibiotics, and the results were interpreted according to the CLSI recommendations (14). antibiotics used in this study belonged to four antimicrobial classes (Beta-lactams, Aminoglycosides, Quinolones, and Macrolides), including Amikacin (30 µg), Cefotaxime (30 µg), Doxycycline (30 µg), Imipenem (10 µg), Piperacillin (30 µg), Trimethoprim/sulfamethoxazole (1.25/23.75 µg),  Ceftriaxone (30 µg), Ceftazidime (30 µg), Nitrofurantoin (100 µg), Ciprofloxacin (5µg),  Norfloxacin (5 µg), Ofloxacin (5 µg), and Nalidixic acid (30 µg), (MAST Group Ltd., UK). E. coli ATCC 25922 was used as a control strain for susceptibility testing. Ciprofloxacin MIC of FQs-R strains was determined by the broth microdilution method and results were interpreted according to the CLSI guidelines (14).
Molecular Detection of Virulence Genes
Genomic DNA of UPEC isolates were extracted using the boiling method. The presence of pap, hly, aer genes among UPEC isolates was investigated by Multiplex PCR using specific primers, as described previously (15). PCR reaction was performed in a final volume of 25µl, containing 3µl template DNA, 2X Taq premix 12.5µl (Ava gene Co, Iran), 2 µl of each primer (Takapouzist Co, Iran), and 3.5 µl ddH2O. The following program was used for amplification of pap, hly, aer genes: initial denaturation at 94˚C for 1 min followed by 30 cycles of denaturation at 94˚C for 1 min, the annealing temperature of 63˚C for the 30s, and extension at 72˚C for 90s, followed by a final extension of 72˚C for 5 min. Detection of PCR products was performed along with a 100 bp DNA ladder on 1.5% agarose gel electrophoresis. The size of amplified fragments of pap, hly, and were 336, 1177, and, 602 bp, respectively. The PCR amplicons were sequenced by Bioneer Company (Korean Biotechnology Co), and the result of the sequences was analyzed by NCBI - BLAST.
The data of this study were analyzed using SPSS software version 16.0 (Chicago, SPSS Inc., IBM, USA). P≤ 0.05 was considered statistically significant.

 
 

Results

Among all urinary tract microbial pathogens, E. coli was the most common isolate, with a frequency of 125 (73.5%) from hospitalized patients. The frequencies of other causative agents of urinary tract infections were Acinetobacter spp. (8.8%), Klebsiella pneumonia (7.1%), Staphylococcus aureus (4.7%), Pseudomonas aeruginosa (3%), and Proteus spp. (2.9%).
The frequencies of resistance to commonly tested antibiotics for E. coli were as follows: Amikacin (26.4%), Cefotaxime (59.2%), Doxycycline (88.0%), Imipenem (55.2%), Piperacillin (54.4%), Trimethoprim/sulfamethoxazole (77.6%), Ceftriaxone (65.6%), Ceftazidime (55.2%), and Nitrofurantoin (25.6%). More than half (54.4%) of the clinical isolates of E. coli showed multi-drug resistant patterns (resistance to three or more classes of antibiotics). The Ciprofloxacin MIC values ranged from 8 to 256 µg/ml (Table 1). Among the 125 UPEC clinical isolates, 111(88.8%) strains were resistant to Nalidixic acid, 89 (71.2%), 88 (70.4%), and 86 (68.8%) isolates were resistant to Ciprofloxacin, Norfloxacin, and Ofloxacin, respectively.

 
Table 1. Ciprofloxacin MIC values

Breakpoint
for Ciprofloxacin
Sensitive Intermediate Resistance
1µg/mL 2µg/mL ≥4 µg/Ml
MIC(µg/mL) 1 256 128 64 32 16 8 4
Isolates NO (%) 36 1 10(11.3) 32(36.3) 23(26.1) 15(17.0) 7(7.9) 1(1.1) 0(0.0)
Total NO (%) 36(28.8) 1(1.12) 88(70.4)

 The results of the amplification of virulence factor genes are shown in Figure 1. The frequency of virulence factors among susceptible and resistant to quinolones and fluoroquinolones UPEC isolates were compared and shown in Table 2. There was a difference between the frequency distribution of virulence genes among resistance and susceptible strains. The prevalence of hly and pap genes in FQ-R and Q-R strains was lower than FQ-S and Q-S strains; however, the prevalence of aer gene was higher in FQ-R and Q-R strains. Statically analysis using the Chi-square test showed a significant difference (P= 0.009) in hly gene distribution between FQ-R and FQ-S strains. Our results also showed a significant difference between the distribution of aer (P= 0.013) and hly (P= 0.042) and Q-R and Q-S isolates (Table 2).

 
Table 2. The frequency of virulence factors among isolates susceptible and resistance to quinolones and fluoroquinolones

pap +
No (%)
pap  -
No (%)
P value aer +
No (%)
aer
No (%)
P value hly +
No (%)
hly
No (%)
P value
Fluoroquinolone Susceptible (n=36) 16(44.44) 20(55.55) 0.102 19(52.77) 17(47.22) 0.352 9(25.0) 27(75.0) 0.009
FluoroquinoloneResistant
(n=89)
26(29.21) 63(70.78) 55(61.79) 34(38.20) 7(7.86) 82(92.13)
Quinolones
Susceptible  (n=14)
6(42.85) 9(64.8) 0.354 4(28.57) 10(71.42) 0.013 4(28.57) 10(71.42) 0.042
Quinolones
Resistant
(n=111)
34(30.6) 77(69.36)   70(63.0) 41(36.93) 11(9.9) 100(90.09)

 
Figure 1. Representative PCR results for virulence genes in UPEC strains. L: Ladder 100bp, Lane 1-5 &7: Clinical isolates (pap: 336 bp, aer: 602 bp, hly: 1177 bp), Lane 6: Positive control, Lane 8: Negative control
Figure 1. Representative PCR results for virulence genes in UPEC strains. L: Ladder 100bp, Lane 1-5 &7: Clinical isolates (pap: 336 bp, aer: 602 bp, hly: 1177 bp), Lane 6: Positive control, Lane 8: Negative control

 
 

Discussion

In this study, we investigated the prevalence of resistance to different antibiotics classes and virulence factors, including hly, pap, and aer genes, among UPEC isolates in Hamadan hospitals. Our study shows uropathogenic E. coli is the most common uropathogenic bacteria with high-level resistance rate to most used antibiotics, especially fluoroquinolones. Low-level resistance to Amikacin and Nitrofurantoin was detected among UPEC isolates. Amikacin and Nitrofurantoin have known as effective antibiotics to treat urinary tract infections and according to our results, we can consider these antibiotics as choice drugs to treat urinary tract infections due to E. coli strains. The high-level resistance to quinolones and fluoroquinolones was also detected, an alarming challenge in treating UTI patient in hospitals. In the previous studies, high-level resistance to most antibiotics as well as fluoroquinolones was reported in UPEC strains in Hamadan hospitals and other regions of Iran (16-19).
Another factor that has been studied in our research was the detection and comparison of urovirulence genes hly, pap, and aer among FQ-R and FQ-S UPEC strains. We focused on the distribution of virulence factors in Q and FQ-resistant UPEC strains as previously described that antimicrobial agents, e.g., Ampicillin, and aminoglycosides, do not significantly correlate with virulence factors (20-23). Here we confirmed the result of previous studies that reported a lower prevalence of α-hemolysin and pap adhesion of FQs-R compared to FQs-S E. coli strains. As previously reported, the prevalence of pap and hly genes was lower in Q-R and FQs-R strains and aer gene was higher in FQs-R UPEC strains (5, 23). Some reports have suggested that quinolone-resistant E. coli have a minor role in upper urinary tract infection and have low VFs than quinolone-susceptible E. coli (24). Oliveria et al., showed the lower prevalence of virulence genes in UPECs, the frequency of virulence genes in 204 strains isolated from urinary tract infections as follows: aer: 41%, sfa: 26%, pap: 25%, hly: 5% (24). Malekzadegan et al. have suggested the association between antibiotic resistance with the isolates harboring certain virulence genes, such as sfa or the presence of iutA, pap GII and PAI marker in UPEC strains and the importance of some urovirulence genes (e.g., iutA, pap GII, and hlyA) as a marker for developing of symptomatic UTIs (18).
Kawamura et al. demonstrated that 113 strains were resistant to quinolones, 64.6% of strains were resistant to quinolones and 81.6% of strains were resistant to fluoroquinolones. The frequency of virulence factors pap gene and hly gene is lower, and the aer gene is higher in FQs-R strains, they also, have compared the prevalence of virulence gene among susceptible and resistance to quinolones, and fluoroquinolones resistant strains, and reported it as follows: in the susceptible strains to quinolones: (hly: 4%, aer: 54.1%,  pap: 62.8%), among resistant strains to quinolones: (pap: 17.8%,  hly: 18.2%, aer: 90%), and the FQs-R isolates: (pap: 5%,  hly: 0%, aer: 75.3%) (25). One of the interesting findings in our study was the detection of more prevalence of aer gene in FQ-R and Q-R strains compared to FQ-S and Q-S strains. Therefore, the aer gene can be considered a crucial virulence factor in resistant UPEC strains. “There is a suggestion that the higher incidence of VFs among susceptible E. coli isolates would depend on their phylogenetic distribution and that VFs would be intrinsic bacterial characteristics”. Some studies have related quinolone resistance and low virulence with phylogenetic origin (22). Horcajada et al. have demonstrated that Q-R UPEC strains from phylogenetic group B2 have fewer virulence factors than Q-S strains. Overall, these observations indicate that the differences in phenotypes, including quinolone resistance and carriage of virulence genes, are associated with the complex context of genetic background (22).


 

Conclusion

In conclusion, our findings indicate a high level of resistance to different classes of antibiotics as well as quinolones and fluoroquinolones in UPEC strains isolated from UTIs in hospitalized patients in Hamadan hospitals. Furthermore, our findings show that aer gene should also be considered a crucial virulence factor in both fluoroquinolone and quinolone-resistant strains. More consideration is required for a rational prescription of antibiotics as well as fluoroquinolones to treat UTIs. Further studies also are required to understand the cause of the difference in the frequency distribution of virulence factors among antibiotic-susceptible and resistant UPEC isolates.

 

Acknowledgment

We would like to thank all members of the microbiology laboratory of Hamadan University of medical science and the staff of the Microbiology laboratory of Sina Farshchian hospital.

 

Funding

This research was supported by the Vice Chancellor for Research & Technology of Hamadan University of Medical Sciences, Hamadan, IRAN (Grant no: 9304101833).

 

Conflicts of Interest

 The authors declare no conflict of interest.


 

Type of Study: Original Research Article | Subject: Medical Bacteriology
Received: 2022/03/14 | Accepted: 2022/06/29 | ePublished: 2022/09/9

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