Pathotypic and phylogenetic studies of urine Escherichia coli isolates from girls <5 years of age in Marvdasht hospital

Zinat Farhadi; Nima Bahador

doi:10.4103/bbrj.bbrj_73_18

ORIGINAL ARTICLE

Year : 2018 | Volume : 2 | Issue : 4 | Page : 281-285

Pathotypic and phylogenetic studies of urine Escherichia coli isolates from girls <5 years of age in Marvdasht hospital

Zinat Farhadi, Nima Bahador
Department of Microbiology, Shiraz Branch, Islamic Azad University, Shiraz, Iran

Date of Submission	08-Jul-2018
Date of Decision	27-Jul-2018
Date of Acceptance	05-Sep-2018
Date of Web Publication	11-Dec-2018

Correspondence Address:
Dr. Nima Bahador
Department of Microbiology, Shiraz Branch, Islamic Azad University, Shiraz
Iran

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_73_18

Abstract

Background: Urinary tract infection includes infection of the kidneys and bladder. Escherichia coli is the most known cause of urinary tract infection. Severity of the infection depends on the host sensitivity and the presence of virulence factors in the bacterium. In this study, antibiotic-resistance patterns and phylogenetic grouping of urine E. coli isolates from girls referred to Shahid Motahari Hospital, Marvdasht, Iran, were investigated. Methods: In this sectional-descriptive study, urine samples of 45 girls under the age of five suspected to urinary tract infection were collected in a period of 3 months. Eleven isolates of E. coli were identified using common morphological and biochemical tests. Antibiotic resistance patterns of isolates were identified. Phylogenetic groups were determined using polymerase chain reaction methods. Results: In this study, the most antibiotic resistance of the isolates was against cephalexin and the least antibiotic resistance was against imipenem (IPM). Besides, all isolates belonged to the phylogenic Group D. Conclusion: Results shows that IPM may be high effective to treat of the patients referred to Shahid Motahari Hospital in Marvdasht. Overall, results of antibiotics susceptibility tests in different area may be different. Therefore, different antibiotics are required to administrate for patients in different regions around the world or even in a country.

Keywords: Antibiotic-resistance pattern, Escherichia coli, phylogenetic group

How to cite this article:
Farhadi Z, Bahador N. Pathotypic and phylogenetic studies of urine Escherichia coli isolates from girls <5 years of age in Marvdasht hospital. Biomed Biotechnol Res J 2018;2:281-5

How to cite this URL:
Farhadi Z, Bahador N. Pathotypic and phylogenetic studies of urine Escherichia coli isolates from girls <5 years of age in Marvdasht hospital. Biomed Biotechnol Res J [serial online] 2018 [cited 2021 Apr 14];2:281-5. Available from: https://www.bmbtrj.org/text.asp?2018/2/4/281/247244

Introduction

Urinary tract infection includes infection of the kidneys and bladder and is considered the second most common bacterial infection after the respiratory tract infection.^[1],[2] Escherichia ^{More Details} coli is the most known cause of urinary tract infection. Severity of the infection depends on the host sensitivity and the presence of virulence factors in the bacterium.^[3],[4]

Emergence of E. coli strains that are able to produce beta-lactamase is increasing around the world. The patterns of antibacterial susceptibility of Enterobacteriaceae family are unpredictable. Therefore, before administrating the antibacterial drugs, susceptibility determination tests should be performed.^[5],[6],[7] To control infections caused by E. coli, beta-lactam antibiotics are often used. However, in recent years, the use of these drugs has been decreased with the increasing of beta-lactamase enzymes.^[8] Antibiotic-resistance patterns of E. coli may change in different geographical regions. Hence, determination of the drug resistance patterns in different geographical regions help to improve the type and dose of the administrated drugs against urinary tract infections.^[9],[10]

Extraintestinal pathogenic and commensal E. coli usually vary in the phylogenetic group and pathogenic characteristics. Phylogenetic grouping of the E. coli isolates can be analysed using polymerase chain reaction (PCR) method to detect genes yjaA and chuA and DNA fragment TspE4.c2 with lengths of 279, 211, and 152-base pair (bp), respectively.^[11],[12] Based on the presence or absence of these genes, E. coli was categorized into major phylogenetic groups including A, B1, B2, or D.^{[11],[12],[13]}

Results of phylogenetic analyzes that were performed around the world on E. coli indicates that a majority of E. coli causing extraintestinal infections belong to Group B2 and in lower rate belong to Group D.^{[3],[14],[15],[16],[17],[18]}

Considering the role of E. coli in generating urinary tract infection in women and girl children, determination of its phylogroups and antibiogram characteristics is critical. Hence, in the present study, it was attempt to determine phylogroups and antibiogram characteristics of E. coli in girls' urine samples. For this reason, the organisms were isolated from urine samples of girls <5 years of age. The most common phylogroup and effective antibiotics that were identified in this study can be used to adjust the type and dose of the administrated antibiotics against urinary tract infections and consequently develop the community health in the studied geographical area.

Methods

Study design and participants

This study is a descriptive, cross-sectional research on girls <5 years of age who referred to Shahid Motahari hospital, Marvdasht, Fars, Iran in a period of 3 months.

Urine sampling

The urine samples were collected and eventually 45 samples were studied. Urine samples were collected in sterile containers and immediately were used for microscopic examination. Fifteen samples were detected to contain more than three to four white blood cells.

Purification and initial identification of isolates

Using a calibrated loop (0.01 ml), urine samples were cultured on eosin-methylene blue [Figure 1]a and MacConkey Agar culture media [Figure 1]b, in sterile conditions. The cultured media were incubated in a period of 18–24 h at 37°C and then were investigated. Cultures in which the number of colonies was ≥10⁵ CFU/ml were considered positive for urinary tract infections. In order to detect E. coli, biochemical tests including oxidase, catalase, triple sugar iron agar, methyl red, Voges–Proskauer, citrate, and indole tests were done^{[5],[19],[20],[21],[22],[23]} [Table 1].

Figure 1: (a) Colonies of Escherichia coli on eosin-methylene blue give a metallic green sheen. (b) Colonies of Escherichia coli on MacConkey agar

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Table 1: Biochemical tests used to identify Escherichia coli

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Antibiotic resistance pattern of isolates

Antibacterial susceptibility test

In the present study, 11 isolates of E. coli were identified. In order to test antibacterial susceptibility, each isolate was separately cultured on the Mueller-Hinton agar. Antibiotic susceptibility to cephalexin (CN) (30 μg), cefotaxime (CTX) (30 μg), cefixime (CFM) (5 μg), imipenem (IPM) (10 μg), ciprofloxacin (CP) (5 μg), and norfloxacin (NOR) (10 μg) was evaluated using disc diffusion assay based on Clinical and Laboratory Standard Institution (CLSI) guidelines.^[24],[25]

Data analysis

The data were analyzed using SPSS version 19 (SPSS Inc., Chicago, IL, USA). Antibiotic pattern of isolates was studied using Kruskal–Wallis analysis. P < 0.05 was considered statistically significant.

Determining the phylogroups of isolates

The phylogenetic groups of the isolates were determined using the polymerase chain reaction (PCR) method.^[11],[12]

DNA extraction

E. coli isolates were incubated in the Luria–Bertani (LB) liquid medium for 18–24 h at 37°C. The cells were washed twice in 1% PBS solution and suspended in 100 μL of distilled water. The suspension was boiled for 10 min at 98°C. Centrifugation resulted in a pellet and an above solution containing DNA. The solution was separated, stored at −20°C, and performed for PCR.^[4]

Polymerase chain reaction amplification

In order to identify TspE4.C₂, yjaA, and chuA genes, a series of primers were used. The list of primers is shown in [Table 2]. The 2x Master Mix Red was obtained from Yekta-Tajhiz Company, Iran. Then, in order to perform the PCR process, the template DNA (2 μL), 2x Master Mix Red (12.5 μL), forward primer (1 μL; 20 pmol in a μL), and reverse primer (1 μL; 20 pmol in a μL) were mixed together. Next, using distilled water, the final volume of the solution was heightened to 25 mL. PCR cycling program was set up as follow: denaturation at 94°C for 30 s, annealing of primers to the DNA template at 59°C for 50 s, extension of the template at 72°C for 70 s, and final extension at 72°C for 7 s. The 3 ml of the reaction products were electrophoresed using the 2% gel agarose for 45 min. The amplified fragments were assessed up to 3 kilobase pair (kb) by means of ladder 100 bp. The enzyme digestion and sequencing were done.^[4]

Table 2: Primers used to identify isolates

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Phylogenetic groupings were done based on the presence or absence of the amplified gene in the PCR products.^{[11],[12],[13]} The proposal was reviewed and approved by Research and Ethical committee of Department of Microbiology, Shiraz Branch, Islamic Azad University Shiraz, Iran. The Ethics Committee Approval number was IR. Shiraz 16330507932044 and date was 2016 -2018.

Results

Among the 45 collected urine samples, 11 samples were identified with positive E. coli. Based on the antibiogram results [Table 3], highest antibiotic resistance was against CN and the least of resistance was against IPM. In the statistical analysis, the effectiveness of seven antibiotics was compared [Table 4]. Considering the calculated means, it appears that CAZ and IPM had a similar activity and compared to other used antibiotics, there was the highest susceptibility against them. The CP, NOR, and CTX can be classified in the next group with moderate activity. CN and CFM can be classified in a group, and the pathogen is resistant against them. The findings about drugs CN, CAZ, and IPM may be more accurate because of their small standard deviation. Therefore, a more definite decision may be made about the antibacterial effects of these antibiotics. The test was statistically significant (P = 0.0003) and confirmed the differences between activities of the used antibiotics. [Figure 2] shows the mean values and confidence interval of the used antibiotics.

Table 3: Antibiotic resistance patterns among Escherichia coli isolates

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Table 4: Effectiveness of seven used antibiotics

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Figure 2: Means of seven antibiotics with their confidence interval

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The PCR test was conducted to identify the presence of the genes yjaA, chuA, and DNA fragment TspE4.C₂ in isolates. [Figure 3], [Figure 4], [Figure 5] show the results of the PCR test on chuA, TspE4.C₂, and yjaA genes, respectively. Considering to [Table 5] and [Figure 3], chuA gene was reported in all samples. The TspE4.C2 was observed in six isolates [Figure 4]. No isolate contained the gene yjaA [Figure 5]. In the figure, lower bands are related to dimer primers. Thus, according to the classification made by Clermont et al.,^[11] all isolates was revealed to belong to group belong to the phylogenetic Group D.

Figure 3: Gel electrophoresis of the polymerase chain reaction products for gene ChuA. Line 1: 100 bp Ladder, Line 2: Negative control, Line 3-13: polymerase chain reaction products of isolates No. 1-11, respectively

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Figure 4: Gel electrophoresis of the polymerase chain reaction products for gene TspE4.C2. Line 1: 100 bp Ladder, Line 2-12: polymerase chain reaction products of isolates No. 1-11, respectively, Line 13: Negative control

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Figure 5: Gel electrophoresis of the polymerase chain reaction products for gene yjaA. Line 1: 100 bp Ladder, Lines 2-12: polymerase chain reaction products of isolates No. 1-11, respectively

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Table 5: Phylogrouping of Escherichia coli considering to the presence of three studied genes

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Discussion and Conclusion

The aim of this study was to isolate urine E. coli isolates from girls <5 years of age and the classification of the bacteria. In this study, out of 850 patients referred to the Shahid Motahari Hospital, 45 patients were girls <5 years of age. Totally, 11 bacterial strains were isolated from these patients. All identified isolates belonged to the phylogenetic Group D.

As it was revealed in the result section, the majority of the isolates were resistant to CFM and CN. The CAZ and IPM functioned similarly and compared to other used antibiotics; there was the most bacterial susceptibility against them. NOR, CP, and CTX can be categorized in a functional category with regard to the mean level of their function. These results may be used to make better decision about the administration of these antibiotics for patients in the investigated geographical area.

In 2002, Woodward et al. determined antimicrobial susceptibility patterns of E. coli in the Intensive Care Unit in Canada on the basis of regional variables. They reported the susceptibility to Meropenem, Cefepime, Ceftriaxone, and CP to be 100%, 98.2%, 93.1%, and 78.3%, respectively. In the present study, rate of susceptibility of the isolates to CP was less than those that were reported by Woodward et al.^[25] In 2004, Kader et al. investigated isolates from patients urine samples. The rates of antibiotic resistance to Amoxicillin, trimethoprim, CP, and CN were reported to be 61%, 47%, 38%, and 31%, respectively.^[26] Bacterial resistances to CP in the present study and above-mentioned study were similar. The bacterial resistance to CN in the present study was observed to be twice as much as the resistance that was observed by Kader et al.^[26] In 2015, Alcántar-Curiel et al. studied isolates from samples of hospitalized patients urine. The rate of antibiotic resistance to CAZ, CTX, IPM, and CP was reported to be 72.8%, 100%, 0%, and 100%, respectively.^[8] The resistance to IPM in the present study and the mentioned study were similar. In the study conducted by Alcántar-Curiel et al., resistance to CAZ was twice as much as the resistance that we observed. Resistance to CTX, observer by Alcántar-Curiel et al. was three times as much as the one in our study. Moreover, some of the isolates in this study were reported to be resistant to several antibiotics that were in line with the research conducted by Iranpour et al. in 2015.^[3]

In the present study, results of phylogenetic grouping showed that all isolates contained gene chuA and lacked gene yjaA. Therefore, they belonged to Group D. In a study conducted by Abdi and Rashki in 2014 in Sistan Province in Iran, isolates separated from patients' urine belonged to Groups B2 (55%), D (22%), A (17%), and B1 (6%)^[4] that is not in line with the finding of this study, because all isolates in this study are in Group D. In 2014, Iranpour et al. studied patients' urinary isolates and reported some phylogenetic groups included B2 (39.9%), E (9.3%), C and Clad1 (6.4%), B1 (5%), D (2.9%), and A (0.7%).^[3] In both studies conducted by Abdi and Rashki and Iranpour et al., primary sets of isolates were larger than our primary sets and isolates were collected from wider areas than our selected area. Therefore, they had the ability to identify other groups. In 2015, Chakraborty et al. showed that in the south of India, the majority of obtained phylogroups belonged to Group D.^[5]

In sum, in this study, the most antibiotic resistance of the isolates was against CN and the least antibiotic resistance was against IPM. Hence, it seems that in treatment of the patients referred to Shahid Motahari Hospital in Marvdasht, IPM would be more effective to be prescribed. Besides, all isolates belonged to the phylogenic Group D that may indicate that this phylogenic group of E. coli is dominant to induce extraintestinal infections in the mentioned geographical region.

Obtaining different antibiotic susceptibility results in similar experiments show that different antibiotic drugs are required to administrate for patients in different regions around the world or even in a country. Human genetic variations, genetic variations of pathogens, and other factors can cause such differences. Therefore, different treatment strategies are used in different regions based on particular features of each area. Moreover, the emergence of resistant strains to some drugs leads to fail treatment, disease progression, and increased mortality. Therefore, systematic studies are required in worldwide to prescribe antibiotics logically. In order to explore antibiotic patterns, other types of antibiotics are recommended to investigate. Finally, different groups of women such as young women having sexual relationship are recommended to include in the future studies.

Acknowledgments

The authors would like to thank the Research Council of Islamic Azad University (Shiraz Branch) for confirmation of this project as a Master of Science thesis in biology (microbiology) (thesis code: 16330507932044).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Grady R. Safety profile of quinolone antibiotics in the pediatric population. Pediatr Infect Dis J 2003;22:1128-32.
2.	Griebling TL. Urologic diseases in America project: Trends in resource use for urinary tract infections in men. J Urol 2005;173:1288-94.
3.	Iranpour D, Hassanpour M, Ansari H, Tajbakhsh S, Khamisipour G, Najafi A, et al. Phylogenetic groups of Escherichia coli strains from patients with urinary tract infection in Iran based on the new Clermont phylotyping method. Biomed Res Int 2015;2015:846219.
4.	Abdi HA, Rashki A. The phylogenetic study of Uropathogenic Escherichia coli strains in Sistan of Iran. J Birjand Univ Med Sci 2014;21:385-59.
5.	Chakraborty A, Saralaya V, Adhikari P, Shenoy S, Baliga S, Hegde A, et al. Characterization of Escherichia coli phylogenetic groups associated with extraintestinal infections in South Indian population. Ann Med Health Sci Res 2015;5:241-6. [PUBMED] [Full text]
6.	Fritzsche M, Ammann RA, Droz S, Bianchetti MG, Aebi C. Changes in antimicrobial resistance of Escherichia coli causing urinary tract infections in hospitalized children. Eur J Clin Microbiol Infect Dis 2005;24:233-5.
7.	Dacher JN, Hitzel A, Avni FE, Vera P. Imaging strategies in pediatric urinary tract infection. Eur Radiol 2005;15:1283-8.
8.	Alcántar-Curiel MD, Alpuche-Aranda CM, Varona-Bobadilla HJ, Gayosso-Vázquez C, Jarillo-Quijada MD, Frías-Mendivil M, et al. Risk factors for extended-spectrum β-lactamases-producing Escherichia coli urinary tract infections in a tertiary hospital. Salud Publica Mex 2015;57:412-8.
9.	Foxman B. Epidemiology of urinary tract infections: Incidence, morbidity, and economic costs. Am J Med 2002;113 Suppl 1A: 5S-13S.
10.	Garcia FJ, Nager AL. Jaundice as an early diagnostic sign of urinary tract infection in infancy. Pediatrics 2002;109:846-51.
11.	Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000;66:4555-8.
12.	Navidinia M, Peerayeh SN, Fallah F, Bakhshi B, Sajadinia RS. Phylogenetic grouping and pathotypic comparison of urine and fecal Escherichia coli isolates from children with urinary tract infection. Braz J Microbiol 2014;45:509-14.
13.	Dhakal BK, Kulesus RR, Mulvey MA. Mechanisms and consequences of bladder cell invasion by uropathogenic Escherichia coli. Eur J Clin Invest 2008;38 Suppl 2:2-11.
14.	Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev 1991;4:80-128.
15.	Johnson JR, Delavari P, Kuskowski M, Stell AL. Phylogenetic distribution of extraintestinal virulence-associated traits in Escherichia coli. J Infect Dis 2001;183:78-88.
16.	Johnson JR, Stell AL, Delavari P. Canine feces as a reservoir of extraintestinal pathogenic Escherichia coli. Infect Immun 2001;69:1306-14.
17.	Johnson JR, Stell AL, Delavari P, Murray AC, Kuskowski M, Gaastra W, et al. Phylogenetic and pathotypic similarities between Escherichia coli isolates from urinary tract infections in dogs and extraintestinal infections in humans. J Infect Dis 2001;183:897-906.
18.	Sabaté M, Moreno E, Pérez T, Andreu A, Prats G. Pathogenicity island markers in commensal and uropathogenic Escherichia coli isolates. Clin Microbiol Infect 2006;12:880-6.
19.	Taghavi K, Fath PM, Hosseinkhani S, Mirzaei M, Behrooj H, Kiani A, et al. Construction and genetic improvement of copper bioreporter Escherichia Coli. Biomed Biotechnol Res J 2018;2:26-30. [Full text]
20.	Sanker P, Satheesan A, Ambika AP, Santhosh VT, Balakrishnan R, Mrithunjayan SK. High moxifloxacin cross – Resistance levels among “newly identifed” ofloxacin – Resistant multidrug – Resistant tuberculosis patients from South India: A ticking bomb or a tricky challenge? Biomed Biotechnol Res J 2017;1:59-64. [Full text]
21.	Maurya AK, Nag VL, Kant S, Sharma A, Gadepalli RS, Kushwaha RA. Recent methods for diagnosis of nontuberculous mycobacteria infections: Relevance in clinical practice. Biomed Biotechnol Res J 2017;1:14-8. [Full text]
22.	Ravinanthanan M, Hegde MN, Shetty V, Kumari S. Critical concentrations of surfactant combination regimens with MTAD™ on vancomycin-sensitive Enterococcus faecalis. Biomed Biotechnol Res J 2017;1:124-8. [Full text]
23.	Kabir F, Muneer S, Kalam A, Sami A, Qureshi S, Hotwani A, et al. Experience with the quantitative Lytagene real-time polymerase chain reaction for the detection of Streptococcus pneumoniae from pediatric whole blood in Pakistan. Biomed Biotechnol Res J 2017;1:71-5. [Full text]
24.	Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement. CLSI Document M100-S23. Wayne, PA: Clinical and Laboratory Standards Institute; 2013.
25.	Woodward DL, Clark CG, Caldeira RA, Ahmed R, Rodgers FG. Verotoxigenic Escherichia coli (VTEC): A major public health threat in Canada. Can J Infect Dis 2002;13:321-30.
26.	Kader AA, Kumar A, Dass SM. Antimicrobial resistance patterns of gram-negative bacteria isolated from urine cultures at a general hospital. Saudi J Kidney Dis Transpl 2004;15:135-9. [PUBMED] [Full text]