Drift of antibiotic resistance genes in pathogenic Enterobacteriaceae: a case study of Escherichia coli

INTRODUCTION

Escherichia coli is a significant opportunistic pathogen in animals, often causing colibacillosis, but its role extends beyond this primary disease. This microorganism is frequently found in diseased animals and humans, particularly in gastrointestinal, obstetric, gynecological, respiratory, and urinary tract infections. E. coli are categorized into 7 pathotypes, including enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC), and shigatoxin-producing (STEC). Additionally, there’s a category for strains that are pathogenic to birds.

Colibacillosis remains a major challenge in animal husbandry, particularly in industrial poultry farming, where it ranks among the most prevalent bacterial infections in birds (is detected in 40–70% of disease cases). A critical complicating factor is the widespread antimicrobial resistance (AMR) in E. coli isolates, with resistance observed against all major classes of antibacterial drugs – in some cases reaching 100% [1][2][3].

The significance of this study is underscored by two critical factors: the substantial economic losses inflicted on livestock production by colibacillosis, and the escalating global prevalence of antimicrobial-resistant E. coli strains associated with this disease. The sensitivity of pathogens to antibiotics can change over time, making ongoing monitoring of antimicrobial resistance crucial, especially for pathogens like E. coli.

This study summarizes the data available in the literature on the resistance dynamics of E. coli isolated from different farm animal species to antibacterial drugs.

Antimicrobial resistance is the ability of microorganisms to resist the effects of antimicrobial agents, including antibiotics. The early years 1945–1963 following Fleming’s 1945 discovery were marked by a belief that the pharmaceutical industry could constantly develop new antibiotics faster than bacteria could develop resistance. Later, the discovery of plasmids, which can transfer resistance genes between bacteria, highlighted a major route for resistance spread and increased concern (1963–1981). This concern grew into a global problem perception from 1981–1992, leading to increased research and funding (1992–2013). Since 2013 the problem continues to worsen due to the emergence of new resistance mechanisms and the increasing spread of resistant microorganisms among populations [4][5].

The use of alternative products like bacteriophages, probiotics, phytobiotics, and antimicrobial peptides is increasingly important in the treatment of bacterial diseases. However, antibacterial drugs are still very widely used for treatment in livestock and poultry farming. Moreover, the use of antibiotics to prevent disease and boost productivity in agriculture is the reason for the creation and accumulation of AMR genetic determinants in Escherichia species. This, in turn, leads to AMR developement and contamination of raw materials and food products [6][7].

The purpose of this study was to summarize the scientific literature data on E. coli AMR resistance dynamics.

MATERIALS AND METHODS

This study was conducted through systematic analysis of literature data on antibiotic resistance patterns in diverse E. coli strains isolated from pathological specimens and biological samples obtained from companion animals, livestock, and poultry.

RESULTS AND DISCUSSION

In 2011 N. N. Shkil presented the results of testing of 21 pathological and biological samples from aborted and stillborn bovine fetuses; 71 samples from newborn calves; 67 samples from calves aged 10 days to 1 month; 47 samples from 1–3-month-old calves and 18 samples from > 3 month old calves. Pathogenic microorganisms were isolated in 32% of samples from animals exhibiting clinical signs of gastrointestinal disease, while respiratory syndrome was observed in 68% of affected calves. Escherichia were isolated from 38% of the samples. Isolation tests were conducted annually from 2001 to 2010. As established by the author, in 2001, 50% of the isolated Escherichia bacteria exhibited high sensitivity to aminoglycosides. In subsequent years, the increased sensitivity to quinolone/fluoroquinolone drugs was established, which peaked in 2006 (66%). By 2007, resistance rates to these agents had reached parity with aminoglycosides. Subsequently, in 2009–2010, bacterial isolates demonstrated significantly greater sensitivity to aminoglycosides (50%) compared to fluoroquinolones (15%). The author observed a consistent inverse relationship in antimicrobial susceptibility patterns, where increased sensitivity to one drug class frequently corresponds with decreased sensitivity to another class within the same temporal period. Furthermore, these susceptibility trends demonstrate cyclical fluctuations, characterized by multi-year periods of increasing sensitivity followed by subsequent declines in subsequent years [8]. E. coli have, apparently, developed a robust mechanism for acquiring resistance to fluoroquinolone antibiotics. Antisense RNA produced by the micF gene does indeed inhibit porin protein synthesis at the translation level. This has a positive effect on the sigma factor content related to multiple stress resistance σ^S in cells. The most significant fluctuations in E. coli resistance are associated with this phenomenon [9].

The analysis of dynamics in sensitivity of E. coli strains from diseased calves to antimicrobial drugs by N. E. Gorkovenko and Yu. A. Makarov revealed resistance to enrofloxacin in 6.5% isolates in 2006; by 2007 their number increased to 36.4%, and in 2010 reached 90.0%. The number of polymyxin-resistant E. coli isolates in 2006 was 23.3%, in 2010 – 75%. Neomycin resistance was revealed in 64.0 and 81.8% in 2006 and 2010, respectively. Thus, an increasing resistance to enrofloxacin, polymyxin and neomycin, which significantly reduces the therapeutic effectiveness of these antibiotics is observed. Tetracycline resistance in 2006–2008 was approximately 70%, in 2009 it decreased to 60%, and in 2010 it reached 100%. Resistance to chloramphenicol in 2006 and 2007 was 60 and 55%, respectively; in 2008–2009 it increased to 80%, and then decreased significantly. Resistance to streptomycin and kanamycin was increasing from 2006 to 2008, an then decreased in 2009. It reached 100% for both drugs in 2010.

Consistent with previous findings, these data reveal cyclical fluctuations in antibacterial resistance patterns. Despite these periodic variations, the overall trend demonstrates progressive escalation of resistance, ultimately culminating in pan-resistance and complete loss of clinical efficacy for some antimicrobial agents [10].

The research presented by D. A. Zhelyabovskaya et al. in 2017 suggests that 71.4% of the studied E. coli strains (O15, O18, O26) isolated from the intestines of newborn calves are polyresistant. These isolates demonstrated resistance to erythromycin (95.2%), tetracycline and penicillin (90.5%), kanamycin (85.7%), ampicillin (76.2%), streptomycin and gentamicin (71.4%) [11].

Analysis by N. M. Al-Hammash and A. V. Ignatenko of E. coli isolates from a dairy farm revealed high resistance prevalence: 94% to benzylpenicillin, erythromycin, and lincomycin; 83% to tetracycline; 61% to ampicillin; 56% to neomycin; 44% to chloramphenicol; 37% to pefloxacin; 33% to polymyxin; and 28% to cephalexin. The isolates demonstrated intermediate susceptibility to neomycin (55%), polymyxin (50%), furadonin (27%), chloramphenicol (16%), and kanamycin (14%). They were susceptible to the following antibacterials: gentamicin (83%), kanamycin (78%), cephalexin (74%), furadonin (72%), pefloxacin (62%), chloramphenicol (39%), neomycin (39%), ampicillin (33%). Isolates showed 100% susceptibility only to ceftriaxone, while absolute resistance was observed to oleandomycin, clindamycin, and oxacillin [12].

In the study by S. N. Zolotukhin et al. 34.8% of E. coli isolates showed susceptibility to gentamicin, 34.2% were resistant and 31.5% were moderately resistant. Ampicillin was active against 57.8% of E. coli isolates, 27.3% were resistant to it, and 14.4% showed moderate resistance. The highest sensitivity was found to ceftriaxone (84.7%), ciprofloxacin (74.2%) and chloramphenicol (60.6%). The test results demonstrated that none of the antibiotics completely inhibited microbial growth (100% inhibition). Most strains were polyresistant to erythromycin, chloramphenicol, streptomycin, tetracycline, neomycin, ampicillin, gentamicin, and penicillin [13].

Between 2016 and 2020, M. E. Ostyakova and I. S. Shul’ga examined the gut microbiota (enterobiocenosis) of newborn calves affected by gastrointestinal disorders. In the course of this work, the resistance of E. coli strains to certain antibacterials was analyzed. The results of the study are the following: isolates showed resistance to benzylpenicillin, ofloxacin, ciprofloxacin and erythromycin. This suggests multidrug resistance of the isolated strains. 91.7% of the isolates were sensitive to polymyxin, 70.6% to cefazolin, 65.5% to streptomycin, 62.5% to amoxicillin / clavulanic acid combination. Therefore, these antibiotics are the drugs of choice for the treatment of intestinal infection caused by E. coli [14].

In the characterization of diarrheagenic E. coli museum strains for pathotypes and AMR genes by Yu. I. Pobolelova and S. P. Yatsentyuk, EPEC E. coli was the most prevalent pathotype, accounting for 29% of cases relative to other pathotypes. The determination relied on fragments of AMR determinants to β-lactam atibiotics (blaTEM, blaSHV genes), florfenicol (floR), chloramphenicol (cat1, cmlA), streptomycin (aadA1 gene), gentamicin (aac3-IV gene). Among the studied strains, 36% had resistance genes to at least one of the antimicrobials under study, and 5 strains demonstrated resistance to two antibiotics simultaneously: 2 strains to chloramphenicol and streptomycin, 2 more to streptomycin and florfenicol, and 1 strain to chloramphenicol and florfenicol. In total, the chloramphenicol resistance genes cat1 and cmlA were identified in 3 and 4 strains, respectively; the streptomycin resistance gene aadA1 was identified in 17 strains, and the florfenicol resistance gene floR was identified in 4 strains. Resistance genes to gentamicin and β-lactams were not detected [15].

Researchers A. A. Golikova and O. A. Manzhurina conducted experiments on susceptibility of E. coli strains isolated from colibacillosis-affected calves to 16 antimicrobials of various pharmacological classes. The authors established the susceptibility of E. coli O20 strain to the following antibiotics: ampicillin, amoxicillin, tetracycline, chloramphenicol, gentamicin, polymyxin, norfloxacin, enrofloxacin and streptomycin. Strain O33 showed sensitivity to the same antimicrobials as O22, but was resistant to gentamicin and streptomycin and sensitive to furazolidone and furadonin. E. coli O137 demonstrated susceptibility to ampicillin, amoxicillin, tetracycline, chloramphenicol, gentamicin, polymyxin, furazolidone, furadonin, norfloxacin, enrofloxacin, and streptomycin [16].

According to E. A. Sazonova, in 2020–2022, E. coli strains tended to develop multidrug resistance to first-generation cephalosporins, penicillins, tetracyclines, macrolides, lincosamides, sulfonamides, and streptomycin. Antimicrobial resistance was reveled in O2, O78, O115, O126, O15, O18, O119, O33, O41, O101, O137, O157:H7 serovariants isolated from swine colibacillosis cases.

At the same time, the E. coli resistance to various antibacterial drugs changed as follows: 44.0% to cephalexin in 2020, 71.4% in 2021, 100.0% in 2022; 29.1% to cefazolin in 2020, 50.0% in 2021, 31.5% in 2022; 6.9, 14.3, 15.3% to ceftriaxone; 73.7, 50.0, 48.7% to amoxicillin; 73.7, 78.7, 81.3% to ampicillin; 80.5, 57.1, 64.3% to tetracycline; 84.5, 100.0, 99.1% to doxycycline; 30.9, 71.4, 72.5% to streptomycin; 83.4, 100.0, 92.3% to erythromycin; 85.7, 92.8, 91.3% to rifampicin; 11.4, 7.1, 10.6% to norfloxanin; 18.3, 7.4, 2.3% to enrofloxacin; 10.3, 7.2, 11.3% to ciprofloxacin in 2020, 2021 and 2022 accordingly. These figures demonstrate that antibiotic-resistant E. coli strains are highly prevalent. Moreover, the antimicrobial resistance increases over time, sometimes (for example, to cephalexin, doxycycline, erythromycin) reaching 100% [17].

Tishchenko A. S. et al. reported that E. coli strains (K99:O141, F41:O26, F41, K88:O157) from calves and piglets with enteric diseases exhibited resistance to amoxiclav, tetracycline, gentamicin, oxacillin, azithromycin, and ceftazidime, highlighting challenges in veterinary antimicrobial therapy. While fluoroquinolones (ciprofloxacin and pefloxacin) demonstrated the highest antibacterial activity among tested agents, the observed intermediate resistance levels diminish their clinical efficacy. Amoxiclav, oxacillin, gentamicin, and azithromycin demonstrated the lowest antibacterial effect [18].

According to I. N. Zhdanova et al., E. coli strains O8, O15, O20, O101, O115, O157 were isolated from calves and adult cattle in the farms of the Perm Krai in 2020–2021. These strains revealed resistance to ampicillin and cefazolin (61.5% for each) and high resistance to ceftriaxone (23.1%), cefoxitin (30.7%), chloramphenicol (61.5%) and tetracycline (79.5%). The isolates were sensitive to imipenem and tobramycin (100%), meropenem (97.4%), amikacin and moxifloxacin (92.3%) [19].

Makavchik S. A. and Sukhinin A. A. studied microorganisms isolated from the milk of mastitic cows in 2021–2022. E. coli cultures were susceptible to neomycin and carbapenems (100%) and resistant to cephalexin (75%), tetracycline (30%), cefotaxime (30%), gentamicin (14%) and ciprofloxacin (7%). The collected data demonstrate a concerning trend of rapidly increasing resistance to cephalosporins, tetracyclines, and aminoglycosides among clinical isolates [20].

The results of studies conducted by A. S. Lokteva et al. demonstrated that E. coli O141 and O33 strains isolated from samples from dead pigs in 2017–2022 turned out to be pan-resistant. Over 90% of pathogenic isolates exhibited polyresistance, with this concerning prevalence persisting throughout the entire study period [21].

The article by I. M. Donnik describes that the majority of bacteria isolated from samples of cervical scrapings, from mammary secretions, nasal and oral swabs of animals, manure, feed and contact surfaces and equipment were resistant to antimicrobials. Escherichia were resistant to rifampicin, semi-synthetic penicillins and tetracyclines (64–67%); approximately 44% of isolates demonstrated low sensitivity to 3–5 antibiotics of different classes; 28% among them to third-generation cephalosporins: ceftriaxone and cefotaxime. The bacteria exhibited high sensitivity to fluoroquinolones: ciprofloxacin, enrofloxacin, and ofloxacin (82%) [22].

Kochkina E. E. and Morozova N. V. assessed antimicrobial resistance in E. coli strains isolated from cats with genitourinary tract disorders. The study revealed that isolates exhibited the sensitivity to cephalosporins (71 ± 10.7%), moderate resistance was observed to aminoglycosides (83.3 ± 8.8%) and synthetic penicillins (66 ± 11.1%) and resistance to macrolides and fluoroquinolones. The sensitivity of the strains to individual antibiotics is presented as follows: all studied cultures were sensitive to cefepime; 83.3 ± 8.8% were sensitive to ceftriaxone and cefazolin; 83.3 ± 8.8% showed moderate resistance to cefotaxime, enrofloxacin, and gentamicin; 66.7 ± 11.1% showed moderate resistance to amoxiclav; and 50 ± 11.8% showed moderate resistance to ciprofloxacin; 16.7 ± 8.8% exhibited moderate resistance to ceftriaxone and cefazolin. The authors report the absolute resistance to tylosin [23].

When assessing antibiotic resistance before using antibacterial drugs, N. N. Muzyka and A. V. Beletskaya isolated E. coli from various bird species. The isolates exhibited sensitivity to gentamicin (19.0%), florfenicol (16.6%), enrofloxacin (14.3%), spectomycin (14.3%), norfloxacin (7.1%), trimethoprim (4.7%), tilmicosin (4.7%), doxycycline (2.4%) and lincomycin (2.4%). Moderate sensitivity was observed for tilmicosin (11.9%), doxycycline, florfenicol, norfloxacin and spectinomycin (4.7% each), trimethoprim, lincomycin and gentamicin (2.4% each). Thus, the overall sensitivity rate to antibacterial drugs was 20% or lower [24].

In 2023, A. S. Krivonogova et al. published the article “Antibiotic resistance of Enterobacteriacea in the microbiomes associated with poultry farming”. In this study, the minimum inhibitory concentration (MIC) was determined for the reference strain E. coli ATCC 25922 against ciprofloxacin, meropenem, cefepime, and ampicillin. The control strains were cultured during 37 days. This period corresponds to the period of broiler raising in commercial poultry farms from hatching to slaughter. The study demonstrated that the reference strain E. coli ATCC 25922 exhibited resistance to ciprofloxacin (MIC = 0.06–0.12 mg/L), meropenem (MIC = 0.12 mg/L), ampicillin (MIC = 2–4 mg/L), and cefepime (MIC = 0.5 mg/L). Under these conditions, antibiotic resistance did not arise because the studied microorganisms lacked active resistance determinants in their genomes and had no opportunity for horizontal gene transfer due to isolation from other microbes.

The sensitivity to antibacterial drugs of microflora isolated from chicken cloacal swabs and litter at different stages of poultry rearing was also analyzed. All E. coli isolates exhibited sensitivity to ampicillin (MIC: 2.0–4.0 mg/L) and ciprofloxacin (MIC: 0.06–0.12 mg/L). Meropenem was active against 74% of isolates at MIC 0.06 mg/L and against all isolates at MIC 0.12 mg/L. 50% of isolates were resistant to cefepime at MIC 0.125 mg/L, while 100% of isolates were susceptible to it at MIC 0.5 mg/L [25].

The results demonstrate that subinhibitory antibiotic concentrations promote pathogen survival and facilitate the development of resistance through vertical gene transfer. These studies demonstrate that carbapenem resistance in pathogenic E. coli serotypes severely limits therapeutic options, underscoring their critical status in clinical management and necessitating improved antimicrobial stewardship and infection control measures.

A group of authors studied the antibiotic sensitivity of pathogenic coliform cultures circulating in a commercial poultry farm in the Omsk Oblast. E. coli O37, O115, and O2 serovariants were isolated from pathological samples from chickens and chicks of different ages in 2018. These isolates exhibited 100% sensitivity to fluoroquinolone formulated into “Triflon” and “Enroflon K” drugs. At the same time, the strains showed absolute resistance to tetracycline, and most strains were resistant to tylosin, gentamicin, doxycycline, and chloramphenicol [26].

Isakova M. N. et al. studied 127 E. coli isolates from bovine mammary secretions and cervical swabs. Phenotypic resistance to rifampicin, semi-synthetic penicillins, and tetracyclines was prevalent among the studied isolates. The cultures showed a weaker resistance to azithromycin, chloramphenicol and tobramycin. Among the tested isolates, 28.46% demonstrated intermediate resistance to third-generation cephalosporins, while 49.02% carried the blaDHA resistance gene [27].

In 2023 M. S. Alexyuk et al. conducted the monitoring of E. coli antimicrobial resistance in the Republic of Kazakhstan. During 3 months, fecal samples from calves with escherichiosis clinical signs were collected on private farms in the Almaty region. 30 E. coli isolates were recovered from the biological samples; 6 of them were presumably identified as O157:H7. Study results revealed that only 4 isolates were susceptible to all tested antibiotic classes, while 7 isolates exhibited resistance to a single antibiotic. The majority demonstrated multidrug resistance (defined as non-susceptibility to ≥ 3 antibiotic classes). Five isolates showed resistance to 7 antibiotic classes, and one of the isolates proved to be resistant to all 8 classes of antibiotics. Most isolates were resistant to ampicillin, tetracycline, gentamicin, florfenicol, and trimethoprim. Resistance to enrofloxacin and amoxicillin / clavulanic acid combination was less prevalent. Almost all isolates were found to be sensitive to colistin. Some strains exhibited intermediate resistance to gentamicin and amoxicillin / clavulanic acid combination [28].

In the study by M. Yu. Syromyatnikov et al. the antimicrobial resistance genes of E. coli isolated from the intestines of 2–5-day-old diarrheic piglets were analyzed. Bioinformatic analysis identified 26 antibiotic resistance genes, including: aminoglycoside resistance (Aac6-Aph2, Aac6-If; StrA, StrB); β-lactam resistance (AmpC1_Ecoli; OXA-10, OXA-14, OXA-16, Penicillin_Binding_Protein_Ecoli, TEM-143, TEM-166, TEM-215, TEM-76, TEM-95); quinolone resistance (QnrB19, QnrB5, QnrD, QnrVC4); sulfonamide resistance (SulI); tetracycline resistance (TetD); trimethoprim resistance (DfrA1, DfrA14, DfrA27); phenicol resistance (CmlA5, CmlA1; FloR). Among 4 detected quinolone resistance genes, QnrD turned out to be prevalent: almost 60% of the genes in this sample. E. coli strains harboring this resistance plasmid are predicted to exhibit broad-spectrum resistance to most antimicrobial agents commonly used in veterinary practice. Among 10 identified β-lactam resistance genes, Penicillin_Binding_Protein_Ecoli was the most prevalent (24%). The prevalence of OXA-16 was 9%, AmpC1_Ecoli – 15%, OXA-10 – 12%, OXA-14 – 11%. The prevalence of TEM-143, TEM-166, TEM-76, and TEM-95 genes was 6% in total and only 1% of the findings were for TEM-215. Among the phenicol resistance genes, CmlA5 (52%) and CmlA1 (44%) were most prevalent. The most common trimethoprim resistance determinant was DfrA14 gene (64%). Among the aminoglycoside-associated genes, StrA (35%) and StrB (31%) were the most prevalent. Tetracycline and sulfonamide resistance genes collectively represented 3% of the total relative abundance of resistance genes. Among the remaining sequences, no individual resistance group exceeded 10% in relative abundance. Specifically, resistance genes for tetracyclines, aminoglycosides, and sulfonamides each accounted for less than 1% of the total [29].

High-throughput sequencing revealed that QnrD, encoding quinolone resistance, was the most prevalent resistance gene identified.

CONCLUSION

The literature review indicates that E. coli frequently demonstrates resistance to: β-lactam antibiotics (particularly benzylpenicillin, penicillin, and cephalexin); aminoglycosides (primarily streptomycin and gentamicin); tetracyclines, macrolides (erythromycin) and lincosamides (lincomycin). The majority of studies demonstrate that E. coli exhibit polyresistance (resistance to ≥ 2 antimicrobial agents), with many strains showing multiresistance (non-susceptibility to ≥ 1 agent from ≥ 3 antimicrobial classes).

Nevertheless, antibiogram results can vary significantly between studies. This can be attributed to the uneven distribution of antibiotic resistance mechanisms within the microbial populations (microbiocenosis) of individual livestock farms. The specific combination of resistance mechanisms in E. coli populations varies between farms and depends on multiple factors: circulating serovars and their genetic backgrounds; spectrum of antibiotics used and their application methods; presence of resistance determinants in farm environments; disinfection efficacy, as subinhibitory disinfectant concentrations may promote microbial adaptation. These factors collectively shape: the specific array of antibiotic resistance genes circulating within the farm’s microbial community; phenotypic resistance mechanisms (including biofilm formation and bacterial persistence) and adaptive resistance (transient survival advantages under antimicrobial pressure). This represents one of the most critical challenges in treating E. coli infections, as the expanding resistance profile increasingly compromises optimal antimicrobial selection.

The literature review revealed key trends in E. coli antimicrobial resistance and confirmed the escalating challenge of antibiotic resistance in animal production systems. Consequently, comprehensive antimicrobial resistance surveillance is required, encompassing both livestock production facilities and their surrounding environments where resistant microorganisms may persist and spread. Monitoring of antimicrobial susceptibility trends will enable evidence-based updates to therapeutic guidelines for E. coli infections in livestock. In addition, it’s crucial to continue the development and implementation of alternative therapies for infectious diseases that don’t rely on antibiotics.

Contribution of the authors: Pimenov N. V. – conceptualization, formulation of key objectives and research goals, critical revision of the manuscript draft with valuable feedback, approval of the final version of the work; Malikova K. P. – conducting research, data collection, and analysis preparation and writing of the manuscript; text revision and editing.

Вклад авторов: Пименов Н. В. – формирование идеи, формулировка ключевых целей и задач, критический пересмотр черновика рукописи с внесением ценных замечаний, утверждение окончательного варианта работы; Маликова К. П. – проведение исследования, сбор и анализ данных, подготовка и редактирование текста работы.