Evaluation of biofilm formation and antibiotic resistance pattern in extended-spectrum β-lactamase-producing escherichia coli strains

Nabi Jomehzadeh; Khadijeh Ahmadi; Zahra Nasiri

doi:10.4103/bbrj.bbrj_270_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 6 | Issue : 2 | Page : 175-179

Evaluation of biofilm formation and antibiotic resistance pattern in extended-spectrum β-lactamase-producing escherichia coli strains

Nabi Jomehzadeh, Khadijeh Ahmadi, Zahra Nasiri
Department of Microbiology, School of Medicine, Abadan University of Medical Sciences, Abadan, Iran

Date of Submission	11-Oct-2021
Date of Acceptance	16-Dec-2021
Date of Web Publication	17-Jun-2022

Correspondence Address:
Nabi Jomehzadeh
Department of Microbiology, School of Medicine, Abadan University of Medical Sciences, Abadan
Iran

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_270_21

Abstract

Background: The increasing prevalence of multidrug-resistant (MDR) Escherichia coli strains, especially extended-spectrum beta-lactamase (ESBL)-producing strains, has become a global health concern. This study was aimed to determine the frequency of bla_CTX-M, bla_TEM, and bla_SHV genes among E. coli isolates from urinary tract infection (UTI) and evaluate their antibiotic resistance pattern. Methods: Totally 98 E. coli isolates were recovered from urine samples of UTI-diagnosed patients. Antibiotic resistance and ESBL production were evaluated by disk diffusion and combined disk methods according to the Clinical Laboratory Standards Institute guidelines. The biofilm formation ability of isolates was assessed using the tube adherence method. ESBL-positive isolates were screened for bla_TEM, bla_CTX-M, and bla_SHV genes by polymerase chain reaction. Results: Among the examined isolates, 25 (25.5%) were detected as ESBL producers and harbored at least one of the studied genes. The bla_CTX-M was the predominant (44%) gene, followed by bla_TEM (24%) and bla_SHV (8%). The isolates revealed variable resistance levels to all antimicrobials, out of which 55.1% were conferred a high resistance rate to different antibiotic classes and considered MDR. Phenotypically, 42.85% of the isolates were biofilm formers, of which the majority (38%) formed moderate biofilms. Conclusions: This study showed that the ESBL-positive isolates were more resistant to some first-line antibiotics, and this highlights the necessity to control and monitor the prescribed antibiotics used for empirical treatment for UTI patients.

Keywords: Biofilm, escherichia coli, extended-spectrum beta-lactamase, urinary tract infection

How to cite this article:
Jomehzadeh N, Ahmadi K, Nasiri Z. Evaluation of biofilm formation and antibiotic resistance pattern in extended-spectrum β-lactamase-producing escherichia coli strains. Biomed Biotechnol Res J 2022;6:175-9

How to cite this URL:
Jomehzadeh N, Ahmadi K, Nasiri Z. Evaluation of biofilm formation and antibiotic resistance pattern in extended-spectrum β-lactamase-producing escherichia coli strains. Biomed Biotechnol Res J [serial online] 2022 [cited 2023 Jun 10];6:175-9. Available from: https://www.bmbtrj.org/text.asp?2022/6/2/175/347711

Introduction

Extended-spectrum beta-lactamase (ESBL)-producing bacteria has conferred resistance to β-lactam antibiotics and can hydrolyze the most commonly used β-lactam antibiotics including penicillins, monobactams, and most cephalosporins, but not cephamycins and carbapenems. Moreover, multidrug resistance is reported frequently among ESBL-producing bacteria, which limits us to prescribe suitable antibiotics against these organisms.^[1] Because plasmids encoding ESBLs are easily transmitted between species, this has become a major threat, especially in hospitalized patients, and often these ESBL-producing organisms being involved in outbreaks.^[2] Escherichia coli strains, as the most frequent etiological agents of community-acquired urinary tract infection (UTI), are often resistant to many of the antibiotics previously prescribed to treat such infections.^[3] Recently, there has been an increase in the emergence of ESBL producing E. coli, presenting a major challenge to healthcare facilities worldwide.^[4] The severity and extent of infections caused by these microorganisms would be further enhanced when the bacteria are organized in biofilms, which constitutes a big challenge for microbiologists and physicians.^[5],[6] The bacteria surrounded by biofilm emerge extremely resistant to antibiotic therapy and are responsible for many persistent infections.^[7] The awareness of antibiotic resistance patterns and biofilm-forming ability of bacterial isolates is most important for providing reliable empirical antibiotic treatment in health-care centers.^[8] There is limited epidemiological information on the prevalence of ESBL phenotypes, along with biofilm formation in E. coli isolates in the southwest of Iran. Hence, we aimed at investigating the antibiotic resistance pattern and biofilm formation among ESBL-producing E. coli isolated from the urine samples of the patients with UTIs in Southwest Iran.

Methods

Ethical Approval

The research was approved the Ethics Committee of Abadan University of Medical Sciences, Abadan, Iran (Ethical Code: IR.ABADANUMS.REC.1399.068).

Study design and bacterial isolates

This cross-sectional study was performed among the patients suspected of UTI referred to clinical laboratories and medical centers affiliated to Abadan University of Medical Sciences, Southwest Iran, from July to December 2020. A total of 98 nonrepetitive E. coli isolates were recovered from midstream urine samples with significant counts (10⁵ CFU/mL or more). Bacterial identification was done using conventional phenotypic and biochemical tests.

Antibiotic susceptibility testing

Antimicrobial susceptibility of isolates was done by Kirby–Bauer disk diffusion method according to the Clinical Laboratory Standards Institute (CLSI) guidelines.^[9] The following antibiotics were used: amoxiclav (AMC; 30 μg), trimethoprim-sulfamethoxazole (SXT; 30 μg), nitrofurantoin (FN; 300 μg), netilmicin (NET; 30 μg), ampicillin (AM; 10 μg), piperacillin (PIP; 100 μg), gentamicin (GM; 10 μg), cefepime (FEP; 30 μg), cefuroxime (CX; 30 μg), ceftazidime (CAZ; 30 μg), cefotaxime (CTX; 30 μg), and cefixime (CFM; 5 μg) (Rosco Diagnostica, Taastrup, Denmark). E. coli ATCC25922 was used as the positive control.

Phenotypic detection of extended-spectrum beta-lactamase producers

All E. coli isolates that were resistant to ceftazidime (30 μg) or cefotaxime (30 μg) were screened for ESBLs using the CLSI criteria. A combination disc test by the disks of ceftazidime (30 μg) and cefotaxime (30 μg) alone and in combination with clavulanic acid (10 μg) was used as a phenotypic confirmatory test for ESBL production. The increase in the zone of inhibition (≥5 mm) around antimicrobial agents in combination with clavulanic acid was considered ESBL positive.^[9] Klebsiella pneumoniae ATCC and E. coli ATCC 25922 700603 were used as positive and negative control strains.

Assessment of biofilm production

The ability of biofilm formation of all isolates was assessed by using the tube adherence method.^[10] A loop full of respective isolates from an overnight culture was added to sterile Trypticase soy broth (Merck, Darmstadt, Germany) supplemented with 1% glucose contained in 5-mL glass tubes. After the incubation period, planktonic growth was visually verified, following which the tubes were emptied gently, washed with phosphate-buffered saline (Sigma-Aldrich; Budapest, Hungary), and dried on a paper towel. The tubes were then stained using a 1-mL solution of 0.1% crystal violet (Sigma Chemical Co., St. Louis, MO, USA) for 3 h at room temperature. Excess stain was discarded, and the tubes were again washed with deionized water three times and patted dry on a paper towel. The test results were interpreted by observing the color of the tube's inner surface. Biofilm formation could be verified by the existence of an adherent film lining at the bottom and on the wall of the glass tubes. The amount of biofilm formed by the isolates was categorized into four categories as negative, weak, moderate, or strong.

Polymerase chain reaction amplification for extended-spectrum beta-lactamase associated genes

The genomic DNA was extracted using a commercial extraction kit (SinaClon BioScience Co., Iran) according to the manufacturer's instructions. The E. coli isolates that had been phenotypically confirmed to be capable of producing ESBL were examined using conventional polymerase chain reaction (PCR) for ESBL-associated genes (bla_SHV, bla_TEM, and bla_CTX-M). The PCR reaction was done in 25 μL volumes containing 12.5 μL of Master Mix RED (Ampliqon, Denmark), 2 μL of extracted DNA, 8.5 μL of double-distilled sterile water, and 1 μL of each specific primer [Table 1].^[11],[12] PCR amplification was done using a C 1000 Bio-Rad Thermal Cycler (Bio-Rad Laboratories, Inc.) for the studied genes according to the following conditions: initial denaturation for 5 min at 94°C, 35 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 56°C, extension for 90 s at 72°C, with a final extension at 72°C for 10 min, and soaked to 4°C. The resulting PCR products were analyzed by 1.5% agarose gel electrophoresis in 0.5 × TBE buffer, stained with ethidium bromide (SinaClon BioScience Co., Iran), and visualized by using ultraviolet light. During PCR, the following reference strains were used as negative and positive controls: E. coli ATCC 25922 and K. pneumonia ATCC 700603.

Table 1: Primers used for extended-spectrum beta-lactamase genes detection

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Statistical analysis

The results were analyzed by using the Statistical Package for Social Sciences (SPSS) version 25.0 (SPSS Inc., Chicago, Illinois, USA).

Results

Out of 98 E. coli strains that were isolated from urine specimens of patients, 76 (77.6%) were from females and 22 (22.4%) from males. The range of the patient's age was from 8 years to 62 years. According to the antibiogram test, the highest percentage of resistance was observed first against ampicillin (87.7%), followed by trimethoprim-sulfamethoxazole (75.5%), cefuroxime (68.3%), and cefixime (65.3%). Additionally, the isolates also showed the lowest resistance to nitrofurantoin (89.8%), gentamicin (69.4%), and cefepime (67.3%) [Table 2]. Our data revealed that 55 (55.1%) isolates were considered multidrug resistant (MDR), as these isolates exhibited resistance to at least one agent in three or more unrelated classes of antibiotics used in this study. With regard to initial ESBL screening, it was determined that 77 (58.2%) isolates had a growth inhibition zone size of ≤22 mm and ≤27 mm with ceftazidime and cefotaxime disks, respectively, and thus initially considered as potential ESBL producers. Nevertheless, the phenotypic combined disk test confirmed that out of all isolates, only 25 (25.5%) were ESBL producers, while 74.5% were designated as ESBLs negative. The resistance percentage of ESBL-producing and non-ESBL-producing isolates is shown in [Figure 1]. For ESBL-producing E. coli strains, the antimicrobial resistance rates were significantly higher than non-ESBL-producing strains for all antibiotics tested. The results of molecular tests showed that all 25 ESBL-producing strains harbored at least one of the studied genes, of which bla_CTX-M gene was the predominant ESBL type (44%) followed by bla_TEM (24%) and bla_SHV (8%), respectively [Table 3]. Additionally, the simultaneous presence of the bla_CTX-M and bla_TEM genes was observed in four (16%) isolates, followed by bla_CTX-M and bla_SHV in one (4%) isolate and all the three bla genes in a single isolate. The biofilm production ability of all strains under the study was evaluated using the tube adherence method. A total of 42 (42.85%) isolates were biofilm formers, of which the majority (38%) formed moderate biofilms. Twelve (28.5%) isolates formed strong biofilms, and 14 (33.33%) isolates formed weak biofilms.

Figure 1: Comparison of antibiotic resistance of extended-spectrum beta-lactamase-positive and extended-spectrum beta-lactamase-negative strains

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Table 2: Antimicrobial resistance pattern of Escherichia coli isolates

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Table 3: Frequency of bla_CTX-M, bla_TEM, and bla_SHV genes in extended-spectrum beta-lactamase-producing isolates (n=25)

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Discussion

At present, the clinical treatment of UTI is becoming a global concern due to the upsurge in infections caused by ESBL-producing E. coli strains that have developed resistance to common antimicrobial agents.^[13] To our knowledge, there is limited epidemiological information regarding the prevalence of ESBL-producing E. coli causing UTIs in Southwest Iran. Accordingly, this study was aimed at evaluating the antibiotic resistance patterns and biofilm formation of 98 E. coli urine isolates and on phenotypic and genotypic detection of the ESBL presence in these isolates. The findings of this study and many previous reports^[14],[15] conducted in this regard indicate a higher frequency of UTIs in female patients compared to the male population. This can be related to a variety of causes, including anatomical features, poor sanitation, and contraceptive usage. The antibiotic susceptibility testing was carried out for all the urine isolates, and it was found that resistance to ampicillin is very high (87.7%). This was in line with several previous local [16,17], and international [18,19] reports and showed that in many situations, treatment with ampicillin alone may be insufficient. Unfortunately, in accordance with recent studies,^[20] a significant prevalence of antibiotics resistance, particularly among MDR isolates (55.1%), was perceived in the majority of the antimicrobials applied to treat UTIs. According to various literature reviews, the worldwide distribution of ESBL-producing Enterobacteriaceae is very diverse and has been reported between 2% and more than 95%.^[21] A recent systematic review and meta-analysis assessed the average prevalence of ESBL-producing E. coli in Iran to also be 43.2%.^[22] In this study, only 25 (25.5%) isolates were phenotypically approved as producers of ESBLs, which is lower than compared with the overall frequency in Iran. Meanwhile, numerous studies in other countries have shown very different frequencies of ESBL-producing E. coli, some of which indicate very alarming statistics, e.g., ESBL rates in Poland,^[23] Turkey,^[24] and South Korea^[25] were reported to be 92.2%, 69.14%, and 84.3%, respectively. As shown in [Figure 1], ESBL-positive isolates demonstrated greater levels of resistance to all antibiotics compared to the ESBL-negative isolates. Many previous studies have also shown that ESBL-producing E. coli isolates are more resistant to some first-line antibiotics,^{[19],[26],[27]} which could be attributed to the extensive and inappropriate use of these drugs in the empirical therapy of UTIs. Molecular investigation of beta-lactamase genes is an important and reliable epidemiological analytic method to determine antimicrobial resistance.^[28] Based on the literature, the prevalence of dominant ESBL genes is very diverse, and it has been found that the most common types are bla_CTX-M, bla_TEM, and bla_SHV. Over the past decade, bla_TEM and bla_SHV genes have been reported as the most common beta-lactamase types. However, in recent years, the role of the bla_CTX-M gene has become increasingly significant, and although its frequency varies in different regions, its overall distribution increased compared to other responsible genes.^[29] In the current study, as expected, bla_CTX-M was the most common ESBL genotype (44%), followed by bla_TEM (24%) and bla_SHV (8%), which was in agreement with the several other earlier reports.^{[19],[28],[30]} Similarly, bla_CTX-M (31.2%) and bla_TEM (27.6%) types were most prevalent in Iran than other β-lactamase-encoding genes.^[22] In this study, 4% of the ESBL-producing E. coli isolates simultaneously harbored all three genes (bla_CTX-M, bla_TEM, and bla_SHV), which was much lower than reported in India (60%).^[31] Additionally, the highest coexistence was related to the concomitant carriage of bla_CTX-M and bla_TEM genes (16%). This concurs with the findings of other researchers from Iran,^[19] Germany,^[28] and Saudi Arabian,^[32] who reported bla_TEM + bla_CTX-M as the most frequent type. Biofilm-producing microorganisms are difficult to treat clinically because these extracellular polysaccharide structures protect bacteria from antibiotics and host defense mechanisms.^[33],[34] We found that 42.85% of E. coli isolates were detected as biofilm producers by the tube adherence method. This rate is lower than what was reported by Shrestha et al.,^[35] which found that 54.1% of uropathogenic E. coli strains to be biofilm formers. The main limitation of our study was the lack of molecular methods to identify biofilm-related genes. Additionally, due to a lack of funds, other ESBL-associated genes were not analyzed.

Conclusions

The results of this study indicated that resistance to antibiotics used for therapy of UTIs caused by ESBL-producing E. coli isolates has a significant rate in this region. The diversity of ESBL genes carried by ESBL-producing isolates was also described, with the bla_CTX-M being the most prevalent. The extensive dissemination of ESBL-producing E. coli could have resulted in a reduction in therapeutic options as well as an increase in curative cost. This highlights the need to control and monitor the use of antibiotics, and so to prevent the spread of more resistant strains, identification methods and appropriate strategies should be considered.

Acknowledgments

This research is taken from a thesis written by Zahra Nasiri, a general medicine student in Abadan University of Medical Sciences, Abadan, Iran. The authors appreciate everyone who has helped this research directly or indirectly.

Financial support and sponsorship

This study was financially supported by research affairs of the Abadan University of Medical Sciences, Abadan, Iran (No: 99T.838).

Conflicts of interest

There are no conflicts of interest.

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