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Table of Contents
Year : 2021  |  Volume : 8  |  Issue : 3  |  Page : 181-189

Differential expression of blaCTX-M-33 with vancomycin/trimethoprim combination in Escherichia coli-producing extended-spectrum β-lactamase isolated from intensive care unit-acquired urinary tract infection

1 Department of Biology, Gachsaran Branch, Islamic Azad University, Gachsaran, Iran
2 Department of Microbiology, Yasooj Branch, Islamic Azad University, Yasooj, Iran

Date of Submission04-Mar-2021
Date of Decision21-Jul-2021
Date of Acceptance24-Jul-2021
Date of Web Publication25-Sep-2021

Correspondence Address:
Dr. Alireza Khodavandi
Department of Biology, Gachsaran Branch, Islamic Azad University, Gachsaran
Dr. Fahimeh Alizadeh
Department of Microbiology, Yasooj Branch, Islamic Azad University, Yasooj
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/iahs.iahs_39_21

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Aim: The aim of this study was to develop combination approach for the treatment of Escherichia coli-producing extended-spectrum β-lactamases (ESBLs) isolated from intensive care unit (ICU)-acquired urinary tract infections (UTIs). Materials and Methods: The observational study was conducted between January 5, 2018- June 5, 2019 to isolate and detect E. coli from UTI patients admitted to ICUs in Shahid Rajaee hospital, Gachsaran, Iran. Morphological, biochemical and molecular methods were conducted to identify E. coli isolates. Phenotypic confirmation of E. coli producing ESBL was performed using ESBL disc diffusion test according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The combination index assay set-up was based on CLSI guidelines to investigate antibacterial susceptibilities to vancomycin alone and in combination with trimethoprim and interpreted with the fractional inhibitory concentration (FIC) index. Eventually, the expression levels of blaCTX-M-33 gene were determined by real-time quantitative reverse transcription–polymerase chain reaction. Results: A total of 90 ICU-acquired UTIs occurred among 255 patients. The combination index assay results showed that vancomycin/trimethoprim combination would be reduced its minimal inhibitory concentration90 value. The vancomycin/trimethoprim combination revealed partial synergistic and indifferent effects (FIC = 0.52–1.50) in the isolates of E. coli-producing ESBL. The results of gene expression analysis indicated that vancomycin/trimethoprim combination caused downregulation of blaCTX-M-33 gene at negligible levels by 55.56–58.82-fold and stopped drug resistant. Conclusion: Vancomycin/trimethoprim combination may diminish resistance in the E. coli-producing ESBL isolated from ICU-acquired UTI patients.

Keywords: Escherichia coli, gene expression, therapeutics, trimethoprim, urinary tract infections, vancomycin

How to cite this article:
Hosseini F, Khodavandi A, Alizadeh F. Differential expression of blaCTX-M-33 with vancomycin/trimethoprim combination in Escherichia coli-producing extended-spectrum β-lactamase isolated from intensive care unit-acquired urinary tract infection. Int Arch Health Sci 2021;8:181-9

How to cite this URL:
Hosseini F, Khodavandi A, Alizadeh F. Differential expression of blaCTX-M-33 with vancomycin/trimethoprim combination in Escherichia coli-producing extended-spectrum β-lactamase isolated from intensive care unit-acquired urinary tract infection. Int Arch Health Sci [serial online] 2021 [cited 2023 Jan 31];8:181-9. Available from: http://www.iahs.kaums.ac.ir/text.asp?2021/8/3/181/326698

  Introduction Top

The most important cause of urinary tract infection (UTI) is the Gram-negative, facultative anaerobic bacterium uropathogenic  Escherichia More Details coli.[1] UTI affects more than half of all women during their lifetime. The costs of UTIs are estimated to be from $2.4 billion to $3.5 billion in the United States.[2] Most UTIs are localized infections, but in some cases, UTI can be complicated by several risk factors that can lead to pyelonephritis, bacteremia, and urosepsis. Nosocomial UTI can lead to life threatening that is a major health concern. UTIs account for 20%–30% of all the intensive care unit (ICU)-acquired infections.[3],[4]

Unfortunately, with the alarming emergence of antibiotic resistance in different bacterial populations, since it undermines the antibiotic effectiveness, it has undoubtedly become a global health threat. Of particular concern, the strains of E. coli are often resistant to most frequently prescribed therapeutic classes of antibiotics.[1],[5] Antibiotic resistance occurs due to changes in the regulation of gene expression, mutations in genes, or horizontal gene transfer, which can affect the bacterial wall construction or produce a number of antibacterial proteins.[6]

Beta-lactam-based antibiotics exhibit the most commonly prescribed therapeutic antimicrobials, and the spread and emergence of resistance to these agents has greatly increased.[5] The mode of action of β-lactam-based antibiotics in inhibition of the growth of bacteria is acylation of active-site serine in essential penicillin-binding proteins (PBPs). The general reaction and main mechanism of antimicrobial resistance in Gram-negative bacteria is commonly assumed that PBPs were the β-lactamase precursors.[7],[8] β-lactamase commonality is the ability to degrade the β-lactam ring. Two structural types of β-lactamases are serine β-lactamases and metallo-β-lactamases. The most important classes of serine-β-lactamases are extended-spectrum β-lactamases (ESBLs) that could be able to hydrolyze the expanded-spectrum cephalosporins and other oxyimino-β-lactams including aztreonam, ceftazidime, and cefotaxime.[5],[8] The CTX type M variants of ESBLs are the main contributor to the multidrug-resistant profile in many Gram-negative bacteria. Clearly, the CTX-M-β-lactamase enzymes are considered ESBLs which belong to the plasmid-borne Ambler's Class A and Bush–Jacoby–Medeiros group 2be. They have been grouped into five clusters based on the amino acid sequence similarities of these enzymes (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25), named after the first described member of each encoded by blaCTX-M genes.[5],[8],[9],[10] The main types of CTX-M enzymes are CTX-M-14 and CTX-M-15 encoded by blaCTX-M-14 and blaCTX-M-15 genes, respectively.[10],[11] Importantly, the blaCTX-M-33 is a point mutant derivative of blaCTX-M-15.[9],[12]

The increasing rate of E. coli-producing ESBL infections has been documented worldwide.[13],[14],[15] Therapy of E. coli-producing ESBL infections is challenging, and the number of effective antimicrobial agents is limited in many countries.[16],[17] Thus, advances in the development of E. coli-producing ESBL infection therapies have focused on the use of antimicrobial agents in combination.[18] This study mainly sought to screen UTI clinical isolates of uropathogenic E. coli for the spread of E. coli-producing ESBL in ICUs. Moreover, we investigated the antimicrobial susceptibilities to vancomycin alone and in combination with trimethoprim on the isolates of E. coli-producing ESBL using the broth microdilution method interpreting the synergistic effects by the fractional inhibitory concentration (FIC) index model. We have also examined the effect of vancomycin/trimethoprim combination on expression levels of blaCTX-M-33 gene in the isolates of E. coli-producing ESBL.

  Materials and Methods Top

An observational study took place at Shahid Rajaee Hospital, Gachsaran, Iran. The current study was conducted on 255 patients admitted to ICU during January 5, 2018, and June 5, 2019. An ICU-acquired UTI was a defined group of patients with a positive urine culture (at least >105 colony-forming units (CFU)/mL of one or two types of bacteria) first identified >48 h after ICU admission. The patients with negative urine culture results before ICU admission were included in the study. Written informed consent was collected from all patients, their family members, or legal guardians. The authors were removed patients' identifying information and replaced with a code, as the samples were collected from patients' data that are only related to patients' age and gender, length of ICU stay, and previous antibiotic use. All ICU patients who died in this study were excluded. This study complied with the ethical guidelines of the Declaration of Helsinki (2008). All protocols, including sampling, sample handling, and analyzing anonymized samples, were approved by the Islamic Azad University (IR.IAU.GACHSARAN.REC.1396.11).

Clinical isolates were identified as E. coli by colony appearance on blood agar and eosin methylene blue, cell appearance, Gram's reaction, catalase and citrate tests, H2S production, indole, motility, methyl red and Voges–Proskauer tests, triple sugar iron agar, and urease tests. The clinical isolates were further distinguished as uropathogenic E. coli by the amplification of a significant virulence factor aerotaxis receptor (aer) gene. The obtained sequences were analyzed against the databases by BlastN with default settings and parameters. The obtained sequence was deposited to GenBank: BankIt-NCBI-NIH (https://www.Ncbi.nlm.nih.gov/WebSub/). E. coli ATCC 25922 was acquired from Iranian Research Organization for Science and Technology. The UTI clinical isolates were grown in Luria-Bertani broth (HiMedia Labs, India) and stored at −80°C with 20% (v/v) glycerol.[14],[19],[20]

The susceptibility of the clinical isolates of E. coli against 12 different types of beta-lactam-based antibiotics including cefixime (CFM; 5 μg/disc), cefotaxime (CTX; 30 μg/disc), ceftriaxone (CRO; 30 μg/disc), ceftazidime (CAZ; 30 μg/disc), ciprofloxacin (CIP, 5 μg/disc), gentamicin (GEN, 10 μg/disc), nalidixic acid (NAL, 30 μg/disc), nitrofurantoin (NIT; 300 μg/disc), trimethoprim (TMP; 5 μg/disc), trimethoprim/sulfamethoxazole (SXT; 1.25/23.75 μg/disc), and vancomycin (VA; 30 μg/disk) (HiMedia Laboratories, Mumbai, India) was measured on Mueller-Hinton agar (MHA; Merck, Research Laboratories, Darmstadt, Germany) for the disc diffusion susceptibility method, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (M02-A12).[21] The susceptibility patterns of clinical isolates of E. coli were analyzed using WHONET 7.12 software program [World Health Organization (WHO) Collaborating Centre for Surveillance of Antimicrobial Resistance, Boston, Massachusetts, USA].

The clinical isolates of E. coli were screened for production of ESBL using ESBL disc diffusion test in accordance with CLSI standards (M100-S28).[22] Disks containing ceftazidime and cefotaxime were used, and MHA plates were incubated at 35°C ± 2°C for 16–18 h. Ceftazidime zone ≤22 mm and cefotaxime zone ≤27 mm were considered indicative of the isolates of E. coli-producing ESBL. The phenotypic identification of the isolates of E. coli-producing ESBL was carried out by both ceftazidime and cefotaxime alone and in combination with clavulanate. Discs containing ceftazidime, ceftazidime-clavulanic acid (CAZ/CA 30/10 μg/disc), cefotaxime, and cefotaxime-clavulanic acid (CTX/CA 30/10 μg/disc) were aseptically placed on an inoculated MHA plates and then incubated at 35°C ± 2°C for 16–18 h. An increase in the inhibition zone diameter of ≥5 mm for either ceftazidime or cefotaxime in combination with clavulanic acid versus either agent alone was indicative of ESBL production.

For combination treatment, 2–8 × 104 CFU/mL of the isolates of E. coli-producing ESBL and two-fold dilution of vancomycin and trimethoprim (range: 0.25–512 μg/mL) alone or in combination were added to the 96-well cell culture plates and then incubated at 35°C. Subsequently, the minimal inhibitory concentration (MIC) assay was conducted by Stat Fax 303 Reader (Awareness Technology, Inc., USA) in accordance with CLSI standards (M07-A10).[23] To calculate the antimicrobial agent interactions, the FIC index model was used.[24] Results were also represented as isobolograms constructed by plotting synergistic concentrations of vancomycin and trimethoprim.

The real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis was based on the procedure previously described.[25] The inoculum of 2–8 × 104 CFU/mL of the isolates of E. coli-producing ESBL (ESBL-74) was treated with vancomycin alone and in combination with trimethoprim 2× and 1× MIC. For each treatment, the total ribonucleic acid was isolated in accordance with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and treated with the RNAse-free DNase I (Fermentas, USA). RNA quality, concentrations, and absorbance ratio were measured by denaturing formaldehyde agarose gel electrophoresis 1.2% (w/v) and NanoDrop spectrophotometer ND-1000 (NanoDrop Technologies Inc., Wilmington, DE), respectively. Total cellular RNA (0.5 μg) was copied into complementary DNA (cDNA) using random hexamer oligonucleotide primer and M-MuLV reverse transcriptase (Fermentas, USA) in accordance with the manufacturer's instructions. Two oligonucleotide primer sequences (blaCTX-M-33: AY238472 and 16S ribosomal RNA (rRNA): LC314477) were generated with the Primer3 software and analyzed by the OligoAnalyzer tool (https://eu.idtdna.com/pages/tools/oligoanalyzer). The E. coli blaCTX-M-33 and 16S ribosomal RNA (16S rRNA) genes were amplified from the synthesized cDNA with the following oligonucleotide primers: blaCTX-M-33, 5'-CGCTCATCAGCACGATAAAG-3' and 5'-CGTCACGCTGTTGTTAGGAA-3', 16SrRNA 5'-ATGTCGATTTAGGCGCTTGT-3' and 5'-CGTTGCATCGAATTAAACCA-3'. The real-time qRT-PCR was performed using SYBR™ Green qPCR Master Mix (Fermentas, EU) in a Bio-Rad MiniOpticon™ system (USA). The cycling conditions included an initial step at 95°C for 5 min, 40 cycles of denaturation at 95°C for 15 s, and subsequently annealing at 60°C for 15 s and extension at 72°C for 15 s. Eventually, the melting reaction occurred at 65°C–95°C/5 s increments. The relative expression levels of blaCTX-M-33 gene were determined by comparative Ct method (2‒ΔCt formula). Normalization was performed using the 16S rRNA gene. Expression difference with statistically significant and a fold change of ≥2-fold or ≤0.5 were reported significant up- or downregulation, respectively.[25]

The results are presented as the three independent experiments ± standard error. Statistical analysis was performed using analysis of variance. The comparison of two means was calculated using Tukey's post hoc test. Differences were considered statistically significant when P < 0.05. Statistical analyses were performed using the SPSS 22.0 for Windows (SPSS Inc. Chicago, IL, USA).

  Results Top

Among the 255 admitted patients to the ICU, 90 clinical isolates of bacteria were isolated and identified as E. coli by standard laboratory methods [Table 1]. One new sequence of E. coli aerotaxis receptor (aer) gene deposited at DDBJ/EMBL/GenBank under the accession number: MN752178. The incidence rate of E. coli was found in 60% (n = 54/90) of clinical isolates of bacteria in female and 40% (n = 36/90) in male patients. This study included patients aged between 9 and 92 years.
Table 1: Morphological, biochemical, and molecular identification of Escherichia coli isolate

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The susceptibility patterns of the clinical isolates of E. coli are displayed in [Table 2]. The isolates of E. coli were confirmed to be resistant to vancomycin with 95% confidence interval = 94.9–100. The incidence of multidrug resistance (MDR) was 57.78% (n = 52/90) of clinical isolates of E. coli. Furthermore, the MDR isolates were resistant to five (n = 1/52; 1.90%), six (n = 4/52; 7.70%), seven (n = 17/52; 32.70%), and eight (n = 30/52; 57.70%) of antibiotics tested. Among clinical isolates identified as MDR E. coli, 100% (n = 52/52) and 48.08% (n = 25/52) were positive for possible extensively drug-resistant and possible pandrug-resistant isolates, respectively. Results showed that a total of 16.67% (n = 15/90) of the isolates of E. coli-producing ESBL were obtained from isolates.
Table 2: Antibiotic susceptibility testing results for clinical isolates of Escherichia coli

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The combination index assay set-up was based on CLSI broth microdilution reference method and the MIC90s and FICs of vancomycin alone and in combination with trimethoprim against the isolates of E. coli-producing ESBL [Table 3]. Among the isolates of E. coli-producing ESBL, 100% (15/15) were resistant to vancomycin (MIC ≥128 μg/mL) and 86.67% (n = 13/15) were resistant to trimethoprim (MIC ≥16 μg/mL). Our data showed that the vancomycin/trimethoprim combination would reduce MIC90 for most of the isolates of E. coli-producing ESBL. In comparison with vancomycin used alone, the MIC90s of vancomycin were reduced 2–64-fold in concomitant use with trimethoprim. Furthermore, by comparison with trimethoprim used alone, the MICs of trimethoprim were reduced 1–2-fold in combination with vancomycin. The FIC index values of all the isolates of E. coli-producing ESBL were partial synergistic and indifferent effects (FIC = 0.52–1.50) in the presence of vancomycin/trimethoprim combination. Isobologram analyses showed partial synergistic and indifferent effects of two-drug combinations between vancomycin and trimethoprim against the isolates of E. coli-producing ESBL.
Table 3: MIC (μg/mL) and FIC values and isobolograms of vancomycin and trimethoprim against isolates of E. coli producing ESBL

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The estimated relative expression levels of blaCTX-M-33 gene revealed significantly different (P < 0.05) in the isolates of E. coli-producing ESBL (ESBL-74) treated with vancomycin alone and in combination with trimethoprim. As seen in [Figure 1], there are no significant differences in expression levels of blaCTX-M-33 gene compared to the untreated control on vancomycin and trimethoprim challenge at both concentrations. Surprisingly, the vancomycin/trimethoprim combination treatment upon 2× and 1× MIC caused downregulation of blaCTX-M-33 gene at negligible expression levels by 58.82- and 55.56-fold, respectively (P ≤ 0.0001).
Figure 1: Gene-expression changes of blaCTX-M-33 in the isolates of Escherichia coli-producing ESBL (ESBL-74) in response to vancomycin alone and in combination with trimethoprim treatment. The experiment was performed thrice, and the graph was plotted using an average value with standard error. The* denotes significant reduction (P ≤ 0.0001) of blaCTX-M-33 gene expression to untreated control

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  Discussion Top

E. coli is the most common cause of UTI and resulted in a considerable human death.[1],[5] The rising prevalence of E. coli-producing ESBL has been attributed to increasing problem of antimicrobial resistance, as well as poor treatment and prevention of UTI.[5],[26] In accordance with scientific papers, an alarming rise in antibiotic resistance to the isolates of E. coli-producing ESBL is highlighted with the results from this study. The use of antimicrobial agents in combination therapies is becoming an increasingly attractive strategy to combat antimicrobial resistance.

To date, there has been increasing interest in examining possible combination antibiotic therapy effect for treatment of E. coli-producing ESBL infections.[17],[18] Evidence of an in vitro study showed promising preliminary result for the synergistic effect of vancomycin/trimethoprim combination against E. coli.[27]

The glycopeptide antibiotic vancomycin is a relatively large size that is greater than the exclusion limit of ß-barrel-shaped porins and thus cannot significantly penetrate the cell membrane and reach its target. Vancomycin alone is not currently used in the treatment of E. coli infections but only when proteins involved in outer membrane biogenesis such as SurA, Dcd, and SmpA are inhibited.[27],[28] Several lines of evidence reported that combination of vancomycin with other antimicrobial molecules can expand the spectrum of useful therapeutics.[28],[29] LaPlante and Sakoulas[30] examined the antibacterial activity of vancomycin alone and in combination with aztreonam or ceftazidime against E. coli and found that vancomycin enhanced the activates of aztreonam or ceftazidime. In the work reported here, we showed synergies between vancomycin and trimethoprim in the isolates of E. coli-producing ESBL. In this regard, Zhou et al.[27] indicated that trimethoprim sensitizes E. coli to vancomycin. A model depicting of synergistic mechanism of vancomycin and trimethoprim is speculated in [Figure 2] (created with BioRender.com). The synergistic of vancomycin in combination with trimethoprim could enhance the entry of vancomycin into E. coli.
Figure 2: A model depicting how synergistic of vancomycin in combination with trimethoprim could affect the outer membrane permeability and to render vancomycin active against Escherichia coli. VAN: Vancomycin, TMP: Trimethoprim, DHF: Dihydrofolate, THF: Tetrahydrofolate

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Trimethoprim is the well-known dihydrofolate reductase inhibitor, the enzyme that catalyzes the last step of folate pathway biosynthesis and thereby is an indirect inhibitor of nucleic acid synthesis.[31] The resistance rate of E. coli to trimethoprim has increased worldwide.[32] Resistance to trimethoprim arises through a variety of mechanisms, including modifications in dihydrofolate reductase, reduced cellular impermeability, modifications in inhibitor, and loss of binding capacity.[32],[33] Evidence showed that the combined effects of trimethoprim and sulfamethoxazole on the viability of E. coli could enhance antibacterial against E. coli.[34] Several other studies have reported the effect of combinations of antimicrobial agents and trimethoprim.[35]

We have checked our results on the expression levels of blaCTX-M-33 gene in the presence of vancomycin alone or in combination with trimethoprim in the isolates of E. coli-producing ESBL. Therefore, our results indicate alterations in the expression levels of blaCTX-M-33 gene. Vancomycin/trimethoprim combination treatment showed downregulation of blaCTX-M-33 gene. Infact, vancomycin/trimethoprim combination inhibited β-lactamases production. Consequently, extracellular-impermeable-vancomycin could be enter to the E. coli producing ESBL cells and may exert a bactericidal effect of trimethoprim. Our findings showed a positive correlation between synergistic effect of vancomycin/trimethoprim combination and expression of blaCTX-M-33 gene. Kjeldsen et al.[36] evaluated the inhibitory growth of cefotaxime against isolates of E. coli-producing CTX-M-1 and investigated the expression of blaCTX-M-1 at mRNA and protein levels. They observed that the mRNA of blaCTX-M-1 and CTX-M-1 protein levels increased in the presence of high cefotaxime concentrations and varied with growth phase. Brochmann et al.[37] investigated the effect of cefotaxime treatment on E. coli-producing CTX-M-15 or CMY-2 β-lactamase to analyze the differences in transcriptomes of β-lactamase genes including blaCMY-2, AmpC, blaTEM-1, blaOXA-1, and blaCTX-M-15. Their results showed that the transcriptional response of cefotaxime -resistant bacteria to cefotaxime were depended on the bacterial species, resistance level and resistance determinant. Singh et al.[38] investigated the expression of 10 β-lactamase genes of ESBL (TEM, SHV, CTX, and OXA), metallo-β-lactamases (NDM-1, IMP, and VIM), and AmpC β-lactamases (ACT, DHA, and CMY) in drug-resistant and sensitive diarrheagenic E. coli treated with 16 antimicrobial agents. A positive correlation was found between the expression of genes and resistant to antibiotics. All the 10 β-lactamase genes involved high levels of expression in resistant isolates of E. coli.

  Conclusion Top

This in vitro study provides preliminary evidence of a potential synergist activity of vancomycin/trimethoprim combination against isolates of E. coli-producing ESBL demonstrating the downregulation of blaCTX-M-33 gene in the isolates of E. coli-producing ESBL treated with vancomycin/trimethoprim combination. Further investigation needs to be conducted to ascertain whether these special events reflect the vancomycin/trimethoprim combination potential for inhibition of β-lactamase gene in ESBL-producing clinical isolates of E. coli. The present research demonstrated that the vancomycin/trimethoprim combination may diminish resistance in the E. coli-producing ESBL and could be of great importance for the development of therapeutic approaches.


We would like to thank the Islamic Azad University of Gachsaran for financial support. The results of the present study are part of the master's thesis (7106271).

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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