Genetic profiling of extended-spectrum β-Lactamase and carbapenemase-producing Escherichia coli O157:H7 from clinical samples among diarrheal patients in Shashemene, Ethiopia

Abstract

Background

Escherichia coli (E. coli) O157:H7, associated with diarrhea, poses a global health risk. In Ethiopia, where diarrhea is common, there is limited knowledge about these resistant strains and a lack of data on Extended-Spectrum β-Lactamase (ESBL) and carbapenemase production. Understanding the prevalence of antimicrobial resistance genes associated with ESBL and carbapenems is crucial for addressing diarrheal disease. This study aimed to investigate the genetic profile of ESBL and carbapenemase coding gene carriage in E. coli O157:H7 from clinical stool samples and evaluate antimicrobial susceptibility patterns.

Methods

A total of twenty-nine bacterial isolates obtained from diarrheal patients were subjected to conventional culture and phenotypic (Kirby Bauer disc diffusion method) testing for antimicrobial resistance. Additionally, screening for the production of ESBL (combined disk method) and carbapenemase (modified carbapenem inactivation method) was conducted. Isolates that tested positive for ESBL and carbapenemase production were further analyzed, targeting five genes (bla_NDM, bla_KPC, bla_CTX−M, bla_TEM, and bla_SHV) associated with ESBL and carbapenemase production. Data analysis was performed using SPSS version 27.0, employing logistic regression and descriptive statistics.

Results

We analyzed a total of 27 isolates that were ESBL-positive and 12 isolates that were found to produce carbapenemase phenotypically. These isolates were obtained from clinical stool samples and (9/27) 33.3% of the isolates were from under five years children, predominantly from urban areas, and those that have contact with domestic animals. Genes coding ESBL were found in (19/27) 70.4% of the isolates, the most predominant being bla_CTX−M and bla_TEM. Eight isolates carried bla_KPC, but none had bla_NDM, while five isolates carried both bla_CTX−M and bla_TEM genes. bla_SHV-carrying isolates showed phenotypic resistance to ampicillin and cephalosporins, while bla_KPC-carrying isolates exhibited resistance to ampicillin, carbapenems, and tetracycline.

Conclusion

This study identifies a significant prevalence of multidrug resistance in E. coli O157:H7, which can be attributed to the presence of resistance genes coding for ESBL and carbapenem production. Key factors contributing to this resistance, such as urban environments, children under the age of five, and domestic animal ownership, have been emphasized. Additionally, this research underscores the urgent need for enhanced surveillance and targeted interventions to address this pressing public health concern.

Introduction

Antibiotic resistance (AMR) in bacterial pathogens poses a significant global public health threat [1]. Escherichia coli (E. coli) O157:H7, a strain known to cause diarrheal illnesses, has exhibited a concerning increase in antibiotic resistance, including extended-spectrum-lactamases (ESBLs) and carbapenemases production [2,3,4]. Even though the administration of antimicrobial is not recommended for treating E. coli O157:H7 infection due to the exacerbation of the clinical condition caused by lysis of the bacteria and subsequent release of Shiga-like toxins [5, 6], The antibiotic resistance pattern of this strain is a matter of public health concern. This is because it has the potential to horizontally transfer resistance genes through conjugation to the normal microbiota or opportunistic pathogens in the gut [7]. This resistance profile significantly limits the effectiveness of conventional antimicrobial, resulting in treatment failures and higher rates of morbidity and mortality [8].

Ethiopia is widely recognized for its substantial burden of diarrheal illnesses, which contribute to elevated rates of morbidity and mortality, particularly among vulnerable populations [9]. However, there exists a dearth of knowledge regarding the prevalence of ESBL and carbapenemase production in E. coli O157:H7 strains within this study setting [10, 11].

Consequently, the primary objective of this investigation is to rectify this knowledge gap by undertaking a comprehensive analysis of the genetic composition of E. coli O157:H7 strains. These strains were sourced from clinical samples obtained from individuals with diarrhea and were tested to ascertain the presence of genes responsible for ESBL and carbapenemase production.

The findings of this study will contribute valuable insights into the presence and prevalence of specific gene responsible for production of ESBL and carbapenemase in E. coli O157:H7 strains in Ethiopia. This data holds significant potential for informing the development of effective infection control measures and promoting antimicrobial stewardship programs. By providing clinicians with scientific evidence, this research can empower them to design treatment regimens based on evidence-based practices, consequently leading to a substantial reduction in the spread of antibiotic-resistant bacteria and improving patient outcomes in cases of diarrheal illnesses.

Methods and materials

Study site and sampling

This study was conducted in Shashemene Town and Shashemene Zuria Woreda under the Ethiopian Cholera Control and Prevention (ECCP) project as section of master thesis project. Eight public primary healthcare facilities (HCFs) found in Shashemene town (Abosto, Awasho, Chebi, Toga, Harbate, Fajogole health centers as well as Melka oda and Shashemene specialized hospitals) were included in this study randomly. Diarrheal stool samples collection and culturing were done from July 2022 to November 2022 and all sample with phenotypically confirmed ESBL and carbapenemase producing E.coli O157:H7 isolates were included in the study.

Bacterial isolation and identification of isolates

All lactose fermenting bacterial isolates suspected to E. coli from MacConkey agar (Condalab, Spain) were identified by running API 20E (Biomerieux, France) biochemical tests. Further identification of the serotype E. coli O157:H7 was done using sorbitol (Condalab, Spain) utilization and O157 antisera (Denka, Japan).

Antimicrobial susceptibility testing

Isolated colonies were phenotypically tested against 15 antimicrobials (Ampicillin (10 µg), Amoxicillin-clavulanate (20/10 µg), Gentamicin (10 µg), Ceftriaxone (30 µg), Cefotaxime (30 µg), Ceftazidime (30 µg), Ciprofloxacin (5 µg), Trimethoprim-Sulfamethoxazole (1.25/23.75 µg), Chloramphenicol (30 µg), Azithromycin (15 µg), Cefuroxime (30 µg), Tetracycline (30 µg), Imipenem(10 µg), Ertapenem (10 µg), and Meropenem(10 µg) (all produced by Condalab, Spain), using the Kirby–Bauer disc diffusion method for the presence of resistance.

Multidrug resistance Bacteria

Based on CLSI guidelines, we have classified E. coli O157:H7 isolates as multidrug resistant (MDR) if they demonstrate non-susceptibility to more than one class of antimicrobials.

ESBL and carbapenemase production

Production of ESBL (combined disk method using cefotaxime, ceftriaxone, and ceftazidime in combination with clavulanic acid) and carbapenemase (modified carbapenem inactivation method using 10-µg meropenem disk) was determined as instructed by CLSI guideline [12]. All ESBL- and carbapenemase-positive isolates were tested for the presence of ESBL and carbapenemase target genes.

Molecular characterization of ESBLs and carbapenemase genes

DNA extraction

The DNA was extracted from fresh colonies of E. coli O157:H7 isolates by the boiling method according to Dashti et al. [13]. Three to five colonies of an overnight growth of each isolate on nutrient agar (Oxoid, UK) were suspended in 500 µL of 1x Tris-acetate-EDTA (TAE) buffer (Sigma, Germany). The suspension was boiled at 94 °C for 15 min in a water bath (Thermo-fisher Scientific, California) and placed in a freezer at − 20 °C for 10 min, then placed at room temperature for one minute and centrifuged at 13,000 g for 5 min. Finally, 150 µL of the supernatant was transferred into a nuclease-free Eppendorf tube and checked for quality and quantity of DNA using gel electrophoresis (After performing gel electrophoresis, the concentration and yield are determined by comparing the intensity of the DNA sample with a DNA quantitation standard. In particular, if a 2 µl sample of undiluted DNA shows a similar intensity to the 100ng standard, the concentration can be calculated as 50ng/µl (obtained by dividing 100ng by 2 µl) [14].) before storage at − 20 °C until analysis.

PCR protocol

ESBL genes (bla_CTX−M group, bla_TEM group, and bla_SHV group) and carbapenem resistance-determining genes (bla_KPC group and bla_NDM group) were detected using conventional PCR method by utilizing primer pairs listed below. (Table 1.)

Table 1 Sequence of primers used in detecting the ESBL and carbapenemase genes

Detection of ESBL genes was performed in one PCR reaction by multiplexing the above three ESBL genes in one reaction tube. Detection of carbapenemase genes was performed in two separate uniplex PCR reactions with two separate reaction tubes. For both ESBL and carbapenemase genes, the PCR was performed in the MJ Mini PTC-1148 thermocycler (Bio-Rad, USA) in a final volume of 25 µL containing 12.5 µL 2 x Hot Star Taq multiplex PCR Master Mix (QIAGEN, Netherland), 1 µL of each primer (2 µM), 2 µL of template DNA, and 8.5 µL of nuclease-free water, in accordance to previously published protocol [15]. The PCR cycling parameters for both reactions were: initial denaturation at 95 °C for 15 min followed by 35 cycles each of denaturation at 94 °C for 30s, annealing at 58 °C for 90s, extension at 72 °C for 90s, and final extension at 72 °C for 10 min. The PCR products were visualized by performing gel-electrophoresis in 1.5% agarose gel after staining in ethidium bromide with the aid of a gel imaging system, GelDoc (Cleaver GelPro). A 100 bp ladder molecular weight marker (Promega-cat no. 15628019) was used to measure the molecular weight of amplified products.

Quality control

A positive American Type Culture Collection (ATCC) was utilized to validate the efficacy of each culture medium employed. Furthermore, during each phase of the confirmatory testing for ESBL and carbapenemases, both positive and negative controls were incorporated. The positive controls included K. pneumoniae ATCC 700,603 for ESBL and K. pneumoniae ATCC 1705 for carbapenemase, while E. coli ATCC 25,922 served as the negative control for both throughout the process.

Data management and statistical analysis

The data were coded and entered into the SPSS version 27.0 software for analysis. Categorical covariates were summarized using frequencies and percentages. Logistic regression and descriptive statistics was employed to investigate the association between antimicrobial resistance and the carried gene with 95% CI (p-value < 0.005).

Results

General characteristics

A total of 29 phenotypically confirmed (16 ESBL, 1 carbapenemase-producing and 11 both ESBL and carbapenemase-producing) isolates were used for this molecular characterization. The majority of the study participants with ESBL and carbapenemase-positive isolates were males (18/29) with male to female ratio of 1.6:1. Nine of the 27 (33.33%) participants with phenotypic ESBL and five of the 12 (41.67%) carbapenemase-producing isolates were < 5 years of age. The majority of participants with isolates that harbored only ESBL genes (15/19) and all 5 isolates that carried both ESBL and carbapenemase genes were from urban areas. All participants infected with ESBL and carbapenemase gene-carrying E. coli o157:H7 owned domestic animals.

ESBL and carbapenemase coding gene carriage

There were 27 isolates phenotypically confirmed to produce ESBL and all these isolates were tested for the presence of the three most common genes of ESBL (bla_CTX−M group, bla_TEM group, and bla_SHV group) (Table 2). Of these ESBL-positive isolates, 19 (70.4%) were confirmed to carry at least one of the ESBL coding genes. Three of these isolates possessed only bla_CTX−M gene, 6 of them carried bla_TEM only, and eight isolates possessed both bla_CTX−M and bla_TEM genes, whereas one isolate had all the three genes. There were eight isolates genotypically verified to be carbapenemase producers, and all of them harbored the bla_KPC gene but not the bla_NDM gene. Furthermore, four isolates had the bla_CTX−M, bla_TEM, and bla_KPC genes, among the five isolates verified to have had both ESBL and carbapenemase coding genes. (Fig. 1.)

Fig. 1

Gel image of the ESBL and carbapenemase genes from E. coli O157:H7 isolates (A-Gel image for ESBL gene PCR amplification, B-Gel image for carbapenemase gene PCR amplification)

ESBL and carbapenemase co-production

Out of the twenty nine ESBL-producing isolates, 10 (34,5%) were both ESBL and carbapenemase producers phenotypically, of which 50% (5/10) isolates were confirmed to have both ESBL and carbapenemase coding genes (80% (4/5) isolates produced bla_CTX−M, bla_TEM, and bla_KPC; but only 20% (1/5) isolate produced bla_TEM, and bla_KPC) (Table 2).

Table 2 Frequency of isolates harboring ESBL and carbapenemase-encoding genes

Antimicrobial resistance status of ESBL and carbapenemase-producing isolates with respect to the presence of specific genes

Antimicrobial resistance was tested against isolates encoding the ESBL (bla_CTX−M, bla_SHV, and bla_TEM) and carbapenemase (bla_KPC) genes. Resistance to ampicillin, amoxicillin with clavulanic acid, and certain carbapenems was observed in a small number of isolates bearing solely the bla_CTX−M; and resistance to ampicillin, amoxicillin with clavulanic acid, cephalosporins, and carbapenems were observed in isolates containing both the bla_CTX−M and bla_TEM genes. The bla_SHV gene-carrying isolates were resistant to ampicillin and cephalosporins but susceptible to carbapenems. All isolates carrying the bla_KPC gene were resistant to ampicillin, carbapenems, and, in many cases, tetracycline. (Fig. 2.)

Fig. 2

Frequencies of Antimicrobial-resistant ESBL and carbapenemase-producing isolates with respect to the presence of specific genes

Discussion

The rise and spread of multidrug-resistant bacteria, particularly those that produce extended-spectrum beta-lactamases (ESBLs) and carbapenemases, pose a danger to global public health. In this work, we wanted to look at the molecular profiles of ESBL and carbapenemase-producing isolates from E. coli O157:H7 isolates obtained from a cohort of participants.

Our findings indicate a high prevalence of ESBL production among the isolates, with nearly one-third demonstrating the ability to produce carbapenemase. This is a critical discovery, as it shows that a significant portion of the E. coli O157 isolates carry and express genes linked to ESBLs and carbapenemases, both of which contribute to antibiotic resistance. This presents a major challenge for treatment, as infections caused by these resistant strains are more difficult to manage.

Among the three groups of genes analyzed in our study, the bla_TEM group was found to be the most prevalent, followed by the bla_CTX−M group. This finding emphasizes the wide range of beta-lactam antibiotic resistance, which includes resistance to penicillins, cephalosporins, and aztreonam. Furthermore, this discovery reveals the potential for horizontal gene transfer of the TEM and CTX-M genes to various bacterial species. This is a matter of great concern, as both genes are frequently located on plasmids, enabling their transfer between different bacteria. This finding is in agreement with the study from Egypt [18] where the bla_TEM gene group was predominant from E. coli in general without selecting for specific strains. However, our finding is in disagreement with reports from studies conducted among E. coli isolates from Portugal [19], Ethiopia [15], and Lebanon [4], where the majority of isolates were shown to carry the bla_CTX−M group gene rather than bla_TEM group as well as India [20], where it was bla_SHV is predominantly detected gene group. This discrepancy could be because of difference in environmental, carrier host, human migration, sanitation and animal feeding habit difference in different areas [21].

Twelve (31.6%) isolates from the total of 38 E. coli O157:H7 were phenotypically confirmed to produce carbapenemase and, therefore, were tested for the presence of the two most common genes of carbapenemase (bla_KPC group and bla_NDM group). Out of these 12 isolates, 8 (66.6%) were confirmed to have the bla_KPC group gene which is in agreement with the finding from the previously mentioned study in Egypt [18]. However, it was different from a study finding India [22] that reported bla_NDM group to be the predominant one rather than bla_KPC gene group. In contrast, none of the isolates in our study was positive for the bla_NDM gene group. Furthermore, some isolates harbored both ESBL and carbapenemase genes, exhibiting different combinations of bla_CTX−M, bla_TEM, and bla_KPC genes.

The difference in distribution of ESBL and carbapenemase coding genes could be due to the influence of several factors, including geographical variations, horizontal gene transfer, antibiotic usage, sanitation and hygiene practices, animal husbandry practices, human migration, and differences in study design [4, 10]. These factors could be possible contributors to the observed discrepancies between our study and others from different regions, highlighting the complex interplay of environmental, healthcare, and social factors in the spread of antibiotic resistance [19, 23, 24]. Understanding these influences is crucial for developing targeted strategies to combat antibiotic resistance in various regions.

Our finding also showed different patterns of phenotypic resistance in association with the presence of specific genes as among isolates bearing bla_CTX−M, bla_TEM, bla_SHV, and bla_KPC. The isolates possessing both bla_CTX−M and bla_TEM genes exhibited a broader resistance profile, including resistance to ampicillin, amoxicillin with clavulanic acid, cephalosporins, and carbapenems. On the other hand, isolates that carry bla_SHV gene showed resistance to ampicillin and cephalosporins but were susceptible to carbapenems. All isolates that harbored bla_KPC gene showed resistance to ampicillin, carbapenems, and often tetracycline. The presence of these specific resistance genes in bacterial isolates can have significant clinical and public health implications. These genes are associated with heavy burden of resistance against commonly used antibiotic, which can complicate both individual treatment options and broader public health strategies. Understanding these genetic markers is vital for tailored treatment strategies, guiding antibiotic prescriptions, and public health interventions to mitigate antibiotic resistance [25].

Another distribution observation in this study was that a high proportion of ESBL and carbapenemase-positive isolates were detected from children under the age of five, where 9 participants carried ESBL-producing isolates while 5 carries carbapenemase-producing isolates. This detection showcases the need for monitoring antibiotic resistance in the pediatric populations since it has consequences for infection management in this vulnerable age group [26, 27].

In terms of geographical distribution, our study found that the majority of individuals with ESBL gene-positive isolates (15 out of 19) were from urban regions, as were all participants with isolates carrying both ESBL and carbapenemase genes. This finding reveals a possible link between urbanization and the occurrence of antibiotic resistance. Cities with high population density and increased antibiotic use may provide favorable conditions for the spread of resistant microorganisms [28, 29].

Furthermore, among people infected with ESBL and carbapenemase gene-carrying E. coli O157:H7, there was a high association between the presence of ESBL genes and the ownership of domestic animals. This observation is consistent with earlier research report that identified animals, particularly livestock, as repositories of antibiotic-resistant bacteria and potential sites of transmission to people [30, 31].

Conclusion and recommendations

Our study underscores the concerning prevalence of multidrug resistant E. coli O157:H7 isolates, with a substantial proportion ESBL and carbapenemase production. The predominance of bla_TEM and bla_KPC genes highlights the genetic diversity that contributes to antimicrobial resistance. The co-occurrence of ESBL and carbapenemase genes, particularly in urban areas and among children under five years of age, indicates a pressing need for area-specific antibiotic stewardship programs. Furthermore, the association between resistant isolates and domestic animal ownership underscores the significance of zoonotic transmission. We would like to advocate for enhanced surveillance of resistance genes, the implementation of stricter antibiotic usage policies, public education on hygiene practices, and targeted interventions for high-risk groups, including pediatric populations and urban communities.

Limitation of the study

We exclusively performed a molecular test that targeted five particular genes. Nevertheless, an epidemiological trace back to ascertain the origin of resistance in positive isolates was not carried out. Regrettably, this can be attributed to insufficient resources.