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ORIGINAL ARTICLE  
Year : 2017  |  Volume : 10  |  Issue : 2  |  Page : 404-408
Comparison of virulence factors fimA, papC, and hly among uropathogenic Escherichia coli isolates producing and nonproducing extended spectrum beta-lactamases


1 Infectious and Tropical Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2 Department of Medical Microbiology and Virology, Faculty of Medical Sciences, Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
3 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

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Date of Web Publication22-Jun-2017
 

   Abstract 

Introduction: The aim of present study was to investigate virulence factors fimA, papC, and hly in clinical isolates of uropathogenic Escherichia coli (UPEC) producing and nonproducing extended spectrum beta-lactamases (ESBLs) in patients with urinary tract infection (UTI). Materials and Methods: A total of 112 Escherichia coli strains isolated from patients with UTI were collected and characterized by biochemical and bacteriological methods. The presence of beta-lactamase enzymes were determined by phenotypic combined disk test. Then, for detection of fimA, papC, and hly virulence genes polymerase chain reaction assay was performed. Moreover, data analysis was conducted by using SPSS 16.0 software. Results: From 112 E. coli strains, 90% were producing ESBLs (resistant to beta-lactam antibiotics) and 10% were none producing ESBLs enzymes by phenotypic method. Out of 32 hly positive strains 28 (87.5%) were ESBLs positive, from 103 fimA positive strains 93 (90.3%) were ESBLs producer and among 52 papC positive strains 47 (90.4%) were detected that have ESBL enzymes by molecular method. Prevalence of ESBL enzymes among fimA, papC, and hly negative strains was closely similar with positive strains and demonstrated high resistance to beta-lactam antibiotics. Conclusion: The results showed the high prevalence of virulence genes hly, papC, and fimA. There is high resistance to beta-lactam antibiotics due to production of ESBLs regardless to prevalence of virulence genes in clinical strains of UPEC. Indeed, there is no significant relationship between presence of virulence gene and ESBL production. Thus, the virulence and drug resistance genes are needed to be examined as a target for therapeutic intervention.

Keywords: Escherichia coli, extended spectrum beta-lactamases (ESBLs), fimA, papC, hyl, urinary tract infection (UTI)

How to cite this article:
Fattahi S, Aghazadeh M, Nahaei MR, Asgharzadeh M, Kafil HS. Comparison of virulence factors fimA, papC, and hly among uropathogenic Escherichia coli isolates producing and nonproducing extended spectrum beta-lactamases. Ann Trop Med Public Health 2017;10:404-8

How to cite this URL:
Fattahi S, Aghazadeh M, Nahaei MR, Asgharzadeh M, Kafil HS. Comparison of virulence factors fimA, papC, and hly among uropathogenic Escherichia coli isolates producing and nonproducing extended spectrum beta-lactamases. Ann Trop Med Public Health [serial online] 2017 [cited 2019 Oct 23];10:404-8. Available from: http://www.atmph.org/text.asp?2017/10/2/404/208732

   Introduction Top


Extended spectrum beta-lactamases (ESBLs) producing  Escherichia More Details coli strains are reported to be the cause of community and hospital acquired infections. E. coli strains causing urinary tract infection (UTI) have capability to produce ESBLs in huge amounts.[1] β-lactam agents, such as penicillins, cephalosporins, monobactams, and carbapenems, are among the most frequently prescribed antibiotics worldwide that the resistance to this class of antibiotics is increasing.[2],[3]

ESBLs are bacterial enzymes that cause the degradation and inactivation of different types of beta-lactam antibiotics by hydrolysis and which result in ineffective compounds.[4],[5] Resistance to extended-spectrum β-lactam antibiotics in Gram-negative bacteria has been emerging rapidly worldwide over the last 2 decades and has been attributed predominantly to the production of β-lactamase enzymes, including plasmid-encoded ESBLs.[6] Indeed, more than 200 different ESBLs have been described to date and these enzymes have been found all over the world in many bacteria, particularly in the family of Enterobacteriaceae.[7] E. coli is responsible for 80-90% of community- acquired UTI and 30-50% of hospital-acquired UTI.[8] E. coli strains especially uropathogenic Escherichia coli (UPEC) have high rate of resistance to antimicrobial agent particularly to beta-lactam antibiotics almost due to production of ESBL enzymes. The increase in ESBL-producing E. coli in UTI is an important clinical concern as this organism are resistant to multiple antimicrobial agents and difficult to treatment, therefore cause of high mortality rate.[7],[8] The severity of UTI is depends on two factors, bacterial virulence and host sensitivity.[9] In order to colonize and establish a UTI, UPEC strains take advantage of set of virulence properties. Several virulence determinants that contribute to the pathogenicity of E. coli in UTI are the product of different genes. These genes are associated with infection in the normal urinary tract of humans including the specific adhesins and toxins.[10] Bacterial adherence to and colonization of the urinary tract by UPEC strains are mediated by the expression of several types of frimbrial and non-fimbrial adhesions like most adhesions. Adherence to uroepithelial cells is an essential stage for the initiation and development of UTI. This process allows bacteria to resist the flushing action of the urine flow and bladder emptying, promoting bacterial persistence, and activation of the host signaling pathways. It has been difficult to precisely define the role of any particular adhesins due to overlapping function. Type I and P fimbriae, the most common fimbriae found in UPEC strains enhance virulence of pathogen and are involved in initial urinary tract colonization. Type 1 fimbriae coded by the fim gene cluster and type P fimbriae coded by pyelonephritis-associated pili (pap) gene are necessary for the recognition and attaching to receptors along the urinary tract.[11] Many UPEC strains produce toxins such as hemolysin, which may be involved in kidney disease. Production of hemolysin causes extensive tissue damage and facilitating bacterial dissemination.[12] Certain UPEC strains have iron sequestration systems to assist in growth of bacteria; others produce a capsule that may help to avoid clearance from the urinary tract by fluids flow.[13],[14]

The aim of this study is to investigate virulence factors fimA, papC, and hly in clinical strains of UPEC producing and nonproducing ESBLs isolated from hospitalized patients with UTI and also survey the relationship between expression of virulence genes and production of ESBL enzymes in these isolates.


   Materials and Methods Top


Study design

In this cross-sectional study, 112 isolates of E. coli strains were collected from central hospital of Northwestern of Iran during July 2014–December 2014. All of isolates associated to urine specimens of hospitalized patients with UTI. Isolated strains were cultured and purified under certain conditions. After identification of the isolates by standard biochemical and bacteriological tests, all of them preserved in Tryptic-Soy Broth (Merck-German) containing 15% glycerol at -20°C.

Phenotypic method for ESBL detection

For detection of ESBL enzymes E. coli strains phenotypic combined disk method (CDM) carried out so that bacterial isolates were inoculated onto Mueller–Hinton agar and ceftazidime (30 μg) and ceftazidime/clavulanic acid (30 μg/10 μg) disks were placed at a center to center distance of at least 30 mm from each other. All plates were incubated at 37ºC for 18 hours. An increase of >5 mm in inhibition zone diameter of ceftazidime/ clavulanic acid disks in comparison to its zone when tested alone and without clavulanic acid confirmed ESBL production.[15]

DNA extraction

For molecular diagnosis, the total DNA of 112 E. coli isolates were extracted by the DNA extraction kit (Bioneer Company, South Korea) according to the manufacturer's protocol for Gram-negative bacteria.[16]

PCR method

The genetic support of the high virulence mediated by virulence factors (VFs) was investigated by polymerase chain reaction (PCR) method about all of the isolates. PCR analysis for hly, fimA, and papC virulence genes was carried out by PCR method using the previously reported specific oligonucleotide primers shown in the [Table 1]. The PCR mixture contained the purified DNA template, forward/reverse primers, and master mix (Cinagen co, Tehran, Iran).[16] The conditions of PCR were applied for amplification of papC and hly genes explained by Yamamoto et al.[17] For amplification of fimA gene, we use PCR conditions ultimately described by Vargas and Gascon and Vila et al.[18],[19] All of samples that had negative results were tested again at least twice to reduce the possibility of false-negative results. Several of strains with positive results in PCR test selected randomly from samples and recovered, and then their sequences were determined and analyzed by using automatic DNA sequencer (Termocycle), in order to perform a quality control for the PCR products obtained.
Table 1 : Primers used for amplification of considered virulence genes using PCR assays

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Statistical analysis

SPSS software version 16 (SPSS 16.0) specific for Windows was employed to achieve statistical analysis. Fisher's exact test was used to evaluate the relationship between the variables. The p-value less than 0.05 (p value <0.05) considered as the significant level.


   Results Top


Among 112 E. coli strains isolated from UTI specimens, 101 (90%) strains were producing ESBL (resistant to beta-lactam antibiotics) and 11 (10%) strains were none producing ESBLs enzymes by phenotypic CDM. Totally from 112 E. coli strains 29%, 92%, and 46% were detected to had hly, fimA, and papC, respectively.

Out of 32 hly+ strains, 28 (87.5%) were ESBLs positive, from 103 fimA+ strains 93 (90.3%) were ESBLs producer, and among 52 papC+ strains 44 (90.4%) were detected that have ESBL enzymes by molecular method. The prevalence of ESBL enzymes among fimA, papC, and hly negative strains was closely similar to positive strains so that among 80 hly - strains 72 (90%) were ESBLs positive and from 9 fimA- strains 8 (88.8%) were ESBL positive and also out of 60 papC - strains 56 (93.3%) produced ESBL enzymes. These results demonstrated that there is no significant relationship between ESBL production and presence of virulence factors and also showed high resistance to beta-lactam antibiotics in UPEC strains isolated from hospitalized patients regardless to expression of virulence genes [Table 2].
Table 2: Relationship between ESBL production and expression of virulence factors in 112 E. coli isolates

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   Discussion Top


E. coli is able to cause a variety of infections, such as UTI, soft tissue infections, bacteremia, and neonatal meningitis. The ability of E. coli to produce variety of infections and having high pathogenicity is due to its virulence factors. Most important virulence factors associated to UPEC strains include various adhesins (such as fimbriae), capsule, toxins (such as hemolysin), protease, and siderophore. These uropathogenic virulence factors are found less frequently among commensal strains of fecal E. coli than UPEC. Virulence factors enable UPEC to colonies selectively on the mucosal uroepithelium, elicit an inflammatory reaction at the site of infection, and eventually proceed from lower urinary tract to renal cavities and cause tissue invasion. virulence factors enable some members of the normal flora to elicit an infection by overcoming the host defense mechanisms.[20],[21] Hemolysin production is mainly associated to pathogenicity of E. coli, especially the more severe forms of infection.[22] Hemolysin production as a VF by urinary isolates of E. coli has been shown by previous workers. It has been suggested that colonization with hemolytic strains of E. coli is more likely to develop into UTIs. Hemolysis, though not essential for establishment of acute pyelonephritis, but may be contribute to tissue injury, pathogen survival in renal parenchyma, and enter into blood stream.[23],[24] There are deferent studies conducted in deferent parts of world which indicated the distribution of various virulence genes (such as fimA, papC, and hly) in UPEC clinical strains and also indicate the role of these VFs in colonization, dissemination, pathogenesis, and biofilm formation of E. coli pathogens especially in hospital-associated isolates.[25]

β-lactam agents, such as penicillins, cephalosporins, monobactams, and carbapenems, are among the most frequently prescribed antibiotics worldwide. In Gram-negative pathogens such as E. coli, β-lactamase enzymes (ESBLs) remain the most important contributing factor to β-lactam antibiotics resistance and their increasing prevalence, as well as their alarming evolution seem to be directly linked to the clinical use of novel class of antibacterial agents.[26] In this study, the prevalence of ESBL was 90%, which could be due to the indiscriminate use of cephalosporins and this causes them to be highly similar to the results of previous studies in India.[27] The presence of beta-lactamase enzymes was not significantly related with papC, fimA, and hly genes in this study, but in the study conducted by Sharma and Bhat[23] hemolysin production was associated to presence of ESBLs. On the contrary, Karisik and Ellington[28] studied several different genes including hly, papC, traT, iutA, and fyuA of ESBL producing E. coli strain that shown the presence of ESBL have a considerable relationship with fyuA and iutA genes . The current study shows UPEC ability to adapt and survive in urinary epithelium by producing virulence factors and also possession of high drug resistance mainly due to ESBL hydrolyzing enzymes. Expression of numerous VFs probably is necessary for causes of various types of infections and high pathogenicity of the strains.[13]

Development of antibiotic resistance is due to the overuse or abuse of antibiotics by patients and also headstrong prescribe of drug by doctors. Unfortunately, because of lack of advanced molecular testing in most clinical laboratories, virulence and drug resistance genes are not detected, so this can be a serious problem for the physician instructions toward proper treatment of patients.[29],[30] Selection of appropriate antibiotics for treatment depends on the results of antibiotic susceptibility testing in addition programmed and proper use of antibiotics by patients under medical supervision requires good policy to limit the emergence and spread of antibiotic resistant strains especially in hospitals.


   Conclusion Top


Results of the current study showed that fimA, papC, and hly genes neither seem to be necessary nor sufficient for the production of ESBL in UPEC isolates. At all, the presence of fimA, papC, and hly virulence genes can be simultaneous with the expression of ESBL genes in UPEC isolates. A heightened awareness of these organisms by clinicians and enhanced testing by laboratories, including molecular surveillance studies, is required to reduce treatment failures to limit the introduction into hospitals and to prevent the spread of these pathogens within the community. Also, the early detection of pathogenic and resistant factors by phenotypic and molecular methods is one of the most important measures to prevent and treatment of serious infectious diseases particularly those caused by resistant strains.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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Correspondence Address:
Hossein Samadi Kafil
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1755-6783.208732

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