https://orcid.org/0000-0001-8952-7295
Figures
Abstract
Background
The incidence of antimicrobial resistance is alarmingly high because it occurs in humans, environment, and animal sectors from a “One Health” viewpoint. The emergence of plasmid-carried mobile colistin-resistance (MCR) genes limits the efficacy of colistin, which is the last-line treatment for multidrug resistance (MDR) against gram-negative infections.
Objectives
The current study aimed to investigate emergence of colistin-resistance (MCR 1–5) genes in E. coli isolated from patients with urinary tract infections (UTIs) in Jordan.
Methods
E. coli (n = 132) were collected from urine specimens. The E. coli isolated from human UTI patients were examined the resistance to colistin based on the presence of MCR (1–5). All isolates were tested against 20 antimicrobials using the standard disk diffusion method. The broth microdilution technique was used to analyze colistin resistance. In addition, the MCR (1–5) genes were detected using multiplex PCR.
Results
Out of the 132 isolates, 1 isolate was colistin-resistant, having a minimum inhibitory concentration of 8 μg/mL and possessing MCR-1. All the E. coli isolates showed high resistance to penicillin (100%), amoxicillin (79.55%), cephalexin (75.76%), nalidixic acid (62.88%), tetracycline (58.33%), or cefepime (53.79).
Conclusion
To our knowledge, this is the first report on the presence of plasmid-coded MCR-1 in E. coli from a patient with UTIs in Jordan. This is a problematic finding because colistin is the last-line drug for the treatment of infections caused by MDR gram-negative bacteria. There is a crucial need to robustly utilize antibiotics to control and prevent the emergence and prevalence of colistin-resistance genes.
Citation: Al Momani WM, Ata N, Maslat AO (2024) Colistin-resistance genes in Escherichia coli isolated from patients with urinary tract infections. PLoS ONE 19(6): e0305431. https://doi.org/10.1371/journal.pone.0305431
Editor: Marwan Osman, Yale University School of Medicine, UNITED STATES
Received: January 30, 2024; Accepted: May 29, 2024; Published: June 12, 2024
Copyright: © 2024 Al Momani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: There are no competing interests.
Introduction
Urinary tract infections (UTIs) caused by antibiotic-resistant gram-negative bacteria (GNB) are the most common bacterial infections encountered by clinicians and are of growing concern owing to limited treatment options [1]. Although other bacteria of the Enterobacteriaceae family can cause urinary tract infections (UTIs) [2], E. coli is the most common etiological agent of UTIs, accounting for up to 80% of all cases [3].
The treatment of UTI is greatly complicated by the emergence of multidrug-resistant (MDR) isolates [4] and the increasing prevalence of MDR E. coli. The issue of MDR is further exacerbated by the fact that pipeline of innovative antibiotics has been exhausted. In such a scenario, colistin has again come to the limelight in the context of clinical use as the World Health Organization (WHO) has categorized it as one of the antibiotics of crucial significance in human clinical settings [5].
Colistin, the last-line antibiotic to treat acute infections caused by MDR GNB [6], is a narrow-spectrum antibiotic that exhibits robust effects against most members of the Enterobacteriaceae family and common non-fermentative GNB [7].
In 2015, researchers in China reported the presence of a plasmid-coded colistin-resistance MCR-1 gene in E. coli. They reported that this gene can be transmitted from one bacterium to another and encodes a phosphoethanolamine transferase that catalyzes the transfer of phosphoethanolamine (a cationic molecule) to lipid A (a key component of LPS), an event that results in altered cell membrane charge, and consequently, the inability of colistin (a cation) to attach to the membrane and induce cell membrane degradation, thereby conferring resistance to colistin [8].
It is thought that MCR -1 was derived from animals and then transferred to humans via horizontal transmission. This hypothesis is underscored by the fact that E. coli isolates harboring MCR-1 have been identified in animal food products [9]. The mindless use of colistin in the veterinary sector, particularly in the absence of stringent laws, has contributed to the global spread of MCR-1 (10% of animal isolates and 0.1–2% of human isolates), as shown in an Egyptian study [10].
Although studies have reported the presence of MCR genes in patients with UTIs in many countries, no study has investigated the prevalence of MCR genes in patients with UTIs in Jordan. In the present study, we aimed to elucidate the occurrence of colistin resistance in E. coli isolates from patients with UTIs in Jordan.
Materials and methods
Sample collection and identification
This study was conducted over a period of 6 months (between 15th of June 2022 to 17th and December 2022) and included 132 E. coli isolates from the urine cultures of patients with UTI. All participants were aged 3–85 years old and all isolates were obtained from Princess Rahma Hospital in Irbid and a clinical diagnostic laboratory in Amman, Jordan. The samples were streaked onto MacConkey, eosin-methylene blue, and blood agar plates (Oxoid, UK). Following incubation at 37°C for 24 h, all isolates were confirmed as E. coli using standard biochemical tests (IMViC and Kligler Iron Agar tests) and molecular identification tests (polymerase chain reaction (PCR) using the Universal Stress Proteins A (UspA) gene having a band size of 884 bp) [11]. E. coli NCTC 12900 UK was used as the positive control. This study was approved by the Ethics Committee of Yarmouk University.
Antimicrobial Susceptibility Testing (AST)
The antibiotic susceptibility profiles of the 132 E. coli isolates were determined using the disk diffusion technique on Mueller–Hinton agar (Oxoid, UK) using a suspension equivalent in turbidity to 0.5 McFarland. The plates were incubated overnight at 37°C. The results were interpreted according to the guidelines recommended by the Clinical Laboratory Standards Institute (CLSI,2017) [12]. E. coli isolates were defined as MDR (resistant to three or more antimicrobial classes), based on the International Expert Proposal for Interim Standards Guidelines [13]. Resistance against the following antibiotics was tested: cephalexin (30 μg), penicillin (10 μg), ciprofloxacin (5 μg), doxycycline (30 μg), aztreonam (30 μg), imipenem (10 μg), gentamycin (10 μg), florfenicol (30 μg), kanamycin (30 μg), tigecycline (15 μg), cefepime (30 μg), amoxicillin-clavulanate (30 μg), cefoxitin (30 μg), sulphamethaxazole-trimethoprim (25 μg), chloramphenicol (30 μg), tetracycline (30 μg), fosfomycin (50 μg), meropenem (10 μg), amoxicillin (10 μg), nalidixic acid (30 μg) (Oxoid, UK).
The minimum inhibitory concentration (MIC) of colistin (colistin sulfate powder, DADvet, Jordan) against the 132 E. coli isolates was determined by microdilution using Muller-Hinton broth (Oxoid, UK). The MIC values ranged from 128 μg/mL to 0.25 μg /mL in a 2-fold dilution order. The clinical breakpoints for colistin resistance were defined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI statements when the MIC value was >2 μg/mL [14]. E. coli NCTC 12900 UK was used as a reference strain for disk diffusion and MIC testing.
Detection of the colistin-resistance genes by multiplex PCR
DNA was extracted using the boiling method [15], briefly, a 300 μL bacterial suspension was prepared from fresh E. coli colonies grown on nutrient agar (Oxoid, UK), the suspension was vortexed and then incubated in a dry bath (Cleaver, UK) at 100°C for 10 min, followed by immediate incubation on ice for another 10 min. After that, samples were placed in a centrifuge (HERMLE, Germany) and centrifuged at full speed for 10 min, the supernatant was stored at -20°C and used as a template for PCR.
All E. coli isolates (n = 132) were screened using multiplex PCR to evaluate the presence of mobile colistin-resistance genes MCR (1–5). The multiplex PCR assay was performed in accordance with the guidelines proposed by the European Centre for Disease Prevention and Control [16]. The reaction was performed in a total volume of 20 μL (4 μL of 5× HOT FIREPol® Blend Master Mix (Solis BioDyne, Estonia), 4 μL DNA template,6 μL nuclease-free water,1.2 μL of each primer pair) (Table 1). PCR amplification was performed on a Thermocycler (BIO-RAD, USA) with an initial DNA denaturation step at 94°C (15 min), followed by 25 cycles beginning with 30 s of denaturation at 94°C, 90 s of primer annealing at 58°C, and 1 min of extension at 72°C. The final extension step was performed at 72°C for 10 min. Amplified products were visualized by electrophoresis on a 2% agarose gel, followed by staining with ethidium bromide and visualization under UV light.
Results and discussion
Isolation and characterization of E. coli
From the 132 urine samples, a total of 132 E. coli were isolated and confirmed. According to sex, 90.2% (n = 119) of the E. coli were isolated from females and 9.8% (n = 13) of the E. coli were isolated from males; according to age, 75% (n = 99) of the E. coli were isolated from adults, and 25% (n = 33) of the E. coli were isolated from pediatric individuals. A total of 132 isolates were confirmed as E. coli by PCR amplification using the Universal Stress Protein A in Escherichia coli (UspA gene), some of which are shown in Fig 1.
Antimicrobial resistance profiles
A total of 132 isolates showed high resistance to penicillin (100%), amoxicillin (79.55%), cephalexin (75.76%), nalidixic acid (62.88%), tetracycline (58.33%), and cefepime (53.79%).,. However, resistance was the lowest for fosfomycin (6.06%), florfenicol (10.61%), and chloramphenicol (15.91%). Readings for each antibiotic were recorded in the following three categories: resistant (R), intermediate (I), and susceptible (S). The antibiotic susceptibilities of the isolates for the 20 antimicrobials used in this study are shown in (Table 2 and Fig 2).
To verify MDR in all the 132 E. coli isolates, the number of antibiotics each isolate exhibited resistance toward (among a total of 20 antibiotics belonging to different classes) was calculated, and the result has been summarized in (Table 3).
Each E. coli isolate was organized according to the number of antibiotic classes against which it showed resistance. Table 4 presents a summary of the resistance profiles of all the 132 E. coli. Of the 132 E. coli isolates, 117 exhibited MDR, i.e., 88.64%.
Colistin MIC
Among the 132 strains isolated, only a single E. coli isolate harbored the MCR-1 gene and showed resistance to colistin (MIC = 8 μg/mL), The remaining isolates were sensitive to colistin with MIC values < 2 μg/mL (Table 5, Fig 2).
Molecular identification of colistin-resistance genes
A total of 132 isolates were screened for the presence of MCR 1, MCR 2, MCR 3, MCR 4, and MCR 5 using multiplex PCR. Our results showed that only 1 of the 132 E. coli isolates carried MCR-1(Fig 3).
A single E. coli isolate harbored the MCR-1 gene
A single E. coli isolate was deemed resistant to colistin based on the MIC value (8 μg/mL) and the multiplex PCR that detected the presence of MCR-1. Summary results for the single E. coli isolate that showed colistin resistance are presented in Table 6.
E. coli was isolated from urine samples (n = 132), out of which 90.2% (n = 119) originated from females with UTIs while 9.8% (n = 13) originated from males with UTIs, moreover and according to age 25% (n = 33) originated from pediatric individuals while 75% (n = 99) originated from adults. UTIs are one of the major sex-related infectious diseases [18]. Structural differences in the female urinary system contribute to the development of UTIs [19]. An assumption to clarify this difference is that anatomical disparities exist, such as a short space between the anus and urethral opening in females or a long urethra in males [18].
E. coli is the most common etiological agent of UTI, accounting for up to 80% of all cases [3, 20]. In this study, MCR-1 was detected in a single isolate (out of 132, 0.76%) derived from the urine sample of a 27-year-old female patient with a UTI. This prevalence rate was approximately similar to that reported in Myanmar (0.23%) [21], and Switzerland (0.12%) [22]. Despite its low spread, the existence of MCR-1 in a clinical isolate in Jordan could be considered a sign that the transmission of resistance genes (mainly MCR-1) from E. coli to humans has been initiated. However, higher prevalence rates have been reported in Egyptian studies with prevalence rates of 22%, [23] 5.6% [24] and 4.5% [25], and another study on UTIs from Bangladesh reported a prevalence rate of 3.52% [26]. The extensive and improper use of antibiotics induces a selective pressure, further complicated by the rapid rise and outbreak of MDR Enterobacteriaceae [24].
This study highlights the prevalence of MDR in urinary E. coli. Finally, 117 of the 132 isolates (88.64%) showed resistance to at least three classes of antibiotics (so as to be called multidrug resistant). This finding is supported by similar studies on UTI showing high rates of resistance to commonly used antibiotics [23]. As a potential justification for such MDR, a high rate of MDR in patients with UTI has been observed as UTI-causing E. coli are known for their capability to form biofilms that aid recurrence, leading to continuous and resistant infection [27].
Colistin has been used internationally as a last-resort antibiotic for infections caused by GNB [9]. Since its first report in China in 2015, the plasmid-carried colistin-resistance gene MCR-1 has been identified in Enterobacteriaceae isolated from animals and humans in different countries [28]. The differences in colistin resistance among different studies can be explained by the number of cases, general situation of patients, geographical regions, different antibiotic regulations, and compliance with infection management measures [24]. The mobile colistin-resistant MCR-1 is more common than other MCR genes, a result that is supported by our study and previous studies [21–25]. In contrast, MCR-2 was reported in E. coli isolated from a patient with a UTI in a study conducted in Bangladesh [26].
All the E. coli isolates were resistant to penicillin. In addition, highest resistance rates, surpassing 50%, were detected for amoxicillin-clavulanate, cephalexin, cefepime, tetracycline, amoxicillin, nalidixic acid, and sulfamethoxazole-trimethoprim. Moreover, high susceptibility rates, exceeding 75%, were detected for florfenicol, tigecycline, chloramphenicol, and fosfomycin. These resistance rates are similar to those reported in Egypt, demonstrating that E. coli isolates from patients with UTIs are highly resistant to amoxicillin-clavulanate, nalidixic acid, sulfamethoxazole-trimethoprim, tetracycline, and cefepime [25].
In this study, the highest resistance rates have been found against β-lactams, a possible reason regarding the excessive resistance rate to those antimicrobial agents is that in Jordan—within the previous few years (2012–2015)—ESBL-producing E. coli (43–54%) have been isolated from UTI patients at a rate that was drastically higher than what was reported in 2009 (10.8%) [29]. The high resistance to these antibiotics may also be due to doctors’ empirical antimicrobial prescription, self-prescription, non-obligation, and drug consumption without permission from the doctor [30].
The detection of MCR-1 in an E. coli isolate from UTI-affected patients was similarly confirmed by multiplex PCR; in a similar manner, MCR-1 was detected in colistin-resistant E. coli isolates from China [31]. A single E. coli isolate that was resistant to colistin was also resistant to multiple classes of antimicrobials but was susceptible to gentamicin, florfenicol, kanamycin, tigecycline, and fosfomycin. Several studies have shown that colistin-resistant isolates are highly resistant to multiple classes of antimicrobials [24].
In the current study, the isolate was deemed resistant to colistin based on the MIC. This E. coli isolate carrying the MCR-1 gene exhibited an MIC value of 8 μg/mL (MIC > 2 μg/mL). These results are similar to those of studies performed in Egypt and Saudi Arabia [25]. Among the 132 strains isolated, a single E. coli isolate harbored the MCR-1 gene and exhibited resistance to colistin (MIC = 8 μg/mL). The remaining isolates were sensitive to colistin with MIC values < 2 μg/mL. These results are similar to studies in Egypt [23, 25]. However, other studies have shown that some isolates that come out negative for the presence of resistance genes in PCR exhibit phenotypic colistin resistance at the MIC [24, 26].
The MIC values for resistant isolates ranged from 2 to 128 μg/mL, and 23 (6.4%) isolates exhibited MIC values of ≥ 8 μg/mL in Jordanian study [32]. In addition, E. coli isolates from broilers have been reported to display the highest resistance to tetracycline 360 (100%), penicillin 359 (99.7%), and amoxicillin 357 (99.2%) [32]. The worldwide increase in the spread of MCR-1 among animal isolates (compared to human clinical isolates) implies that animals are the probable sources of MCR-1 present in humans. Furthermore, misuse of colistin in agriculture and poultry may be the principal reason for the elevated prevalence of MCR-1 in bacteria isolated from animals and animal yields [32, 33]. In veterinary science, colistin is used for various purposes, including the prophylaxis and treatment of enteric infections, in addition to its use as a dietary supplement in poultry farms to prevent infections caused by pathogenic bacteria [23].
This study provides data on the antimicrobial resistance patterns of E. coli isolated from patients with UTIs. We concentrated on UTIs because they still represent a major source of infection in humans [22]. E. coli from community-received infections involve the interplay between the environment and hospitals, playing a possible role as an exchange platform for MCR-like genes within the environment [22], and E. coli is the most common member of Enterobacteriaceae isolated from clinical samples [24].
The current study had several fundamental limitations, such as the cross-sectional design without future follow-up due to resource limitations, and the fact that recurrent infections were not sequestered from first-time infections. The data in the current study show a worrisome spread of MCR-1-carrying colistin-resistant E. coli, as found in previous studies in Jordan in humans and broilers [32].
These results may reflect the prevalence of colistin-resistant E. coli in Jordan or the silent spread of MCR-1 in humans. Furthermore, analysis of the genetic data of MCR-1-positive strains could help us understand the origin of this gene. The presence of MCR in this study indicates a massive public health issue; this is especially important as colistin antibiotics are the last-line drugs for infection treatment. MCR genes are carried by plasmids and can be spread via horizontal gene transfer to other commensal and pathogenic bacteria [34, 35]. A coordinated strategy for determining MCR-1 dissemination is needed to restrict the spread of multidrug-resistant isolates among patients [24]. More stringent rules must be enforced to halt the further dissemination of colistin-resistant genes [26].
Conclusions
Colistin is considered one of the last lines of therapy that had been used to treat extreme infections caused by MDR pathogens. The development of plasmid-mediated colistin resistance in E. coli poses a serious problem due to its high potential for spreading in medical settings. MCR-1 has been reported in most continents and has been observed in numerous bacterial isolates, especially E. coli, from animals, humans, and the environment. In Jordan, colistin-resistant E. coli harboring MCR-1 was recorded for the first time in patients with UTIs. This highlights the potential health risks that plasmid-carried colistin-resistant genes in E. coli can be detrimental to millions of humans in Jordan. In addition, guidelines should be established on the use of colistin in the human and animal sectors to ensure the success of the therapy and prevent the spread of these resistance genes.
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