Third-Generation Cephalosporin-Resistant Escherichia coli Isolates Belonging to High-Risk Clones Obtained from Fresh Pork Meat in La Plata City, Argentina ^†

Abstract

High-risk clones represent a major concern, as they are very efficient vehicles for mobile genetic elements carrying antimicrobial resistance genes and therefore promote their spread, especially if they confer resistance to cefotaxime, ciprofloxacin, and fosfomycin, included within the highest-priority, critically important antimicrobial agents (HPCIA). Between February 2022 and April 2024, 138 pork samples were obtained from 46 butcher shops in La Plata, Buenos Aires, Argentina. A total of 102 HPCIA-resistant E. coli were isolated. Eighty-five HPCIA-resistant E. coli were selected for whole-genome sequencing. Of these, 27 belonged to 9 clones described as high risk: ST101 (n = 5), ST10 (n = 4), ST48 (n = 4), ST744 (n = 4), ST23 (n = 3), ST58 (n = 2), ST88 (n = 2), ST117 (n = 2), and ST410 (n = 1). Twelve of them were third-generation cephalosporin-resistant. Resistance was mediated by bla_CTX-M-55 (n = 7), bla_CTX-M-14 (n = 4), bla_CTX-M-8 (n = 1), and bla_CMY-2 (n = 1). This study highlights the importance of food products and the food production chain as reservoirs of high-risk clones and resistance genes of epidemiological relevance to public health.

1. Introduction

Antimicrobial resistance (AMR), which arises when bacteria evolve and render antibiotics less effective, has become one of the most pressing public health challenges of the 21st century [1]. Escherichia coli stands out due to its ubiquity, pathogenic potential, and its role as a reservoir and vector for resistance genes across humans, animals, and the environment [2].

E. coli is widely distributed in livestock and is commonly isolated from meats, particularly pork and poultry, which are key components of the human diet [3]. The use of antimicrobials in food animal production, often aimed at promoting growth and disease prevention, has accelerated the development and spread of antimicrobial-resistant strains. During slaughter processing and handling, zoonotic pathogens, including AMR E. coli, may contaminate meat and enter the food chain [4].

Considering the One Health concept, the World Health Organisation (WHO) recently redefined the classification of antimicrobials based on their relevance to human and veterinary medicine. Third-generation cephalosporins, authorised for both human and animal use, are now designated as the highest-priority critically important antimicrobial agents [5]. In parallel, third-generation cephalosporin-resistant (3GC-R) E. coli has been included in the WHO’s Bacterial Priority Pathogens list since 2024 [6].

Based on genetic markers, E. coli can be divided into eight major phylogenetic groups: A, B1, B2, C, D, E, F, and clade I [7,8]. Among them, the ST131 clone, belonging to phylogroup B2, has emerged as the predominant high-risk clone worldwide [7]. More specifically, high-risk clones are defined as strains that are globally distributed, harbour multiple resistance determinants, effectively colonise and persist in the host, transmit efficiently between hosts, and exhibit increased pathogenicity and fitness, often leading to severe or recurrent infections [9].

Given this context, this study aimed to determine the presence of high-risk clones among the highest-priority, critically important antimicrobial agents (HPCIA)-resistant E. coli isolated from fresh pork meat at butcher shops in La Plata, Buenos Aires, Argentina.

2. Materials and Methods

2.1. Retail Markets Sampling

Between February 2022 and April 2024, fresh pork meat samples were purchased from 46 randomly selected retail markets in La Plata, Buenos Aires, Argentina. A total of 138 pork samples were collected. The meat cuts principally obtained directly from the counter in each butcher shop were pork chops, shoulder, and ham.

2.2. Sample Processing

Briefly, 25 g of each sample was mixed with 225 mL of buffered peptone water (Britania, Buenos Aires, Argentina) and then incubated overnight at 37 °C. Enriched culture (30 µL) was inoculated on MacConkey agar (Britania) plates added with 4 µm/mL of cefotaxime (Sigma-Aldrich, Saint Louis, MO, USA) or 1 µm/mL of ciprofloxacin (HCL salt) (Sigma-Aldrich) and eosin methylene blue agar (Britania) plates with a fosfomycin disc (200 µg) (Britania), followed by incubation at 37 °C for 18 h. One colony consistent in appearance with E. coli was selected per plate; if colonies had different morphologies, up to two colonies were picked. Suspected colonies were subcultured into tryptein soy agar (Britania) and identified by traditional biochemical tests. Those colonies that were confirmed to be E. coli were preserved at −20 °C in brain-heart broth (Britania) with 30% glycerol (Anedra, Buenos Aires, Argentina).

2.3. Whole Genome Sequencing and Bioinformatics

Whole genome sequencing (WGS) of 85 HPCIA-resistant E. coli isolates randomly selected was performed on Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) using a 250-base-pair paired-end protocol. The results were analysed on Galaxy platform version 24.2.

3. Results

Of the total samples processed, at least one HPCIA-resistant E. coli was obtained in 52.17% (72/138). A total of 102 HPCIA-resistant E. coli were isolated.

Of the total 85 strains of HPCIA-resistant E. coli selected for sequencing, 31 of them, harboured bla_CTX-M variant genes alone or in combination with other genes that confer resistance to fluoroquinolones and/or fosfomycin, respectively (14 E. coli harbour bla_CTX-M/qnr, 7 bla_CTX-M/fos/qnr, 2 bla_CTX-M/fos, and 8 bla_CTX-M). Of these, 27 belonged to nine clones described as high risk (Figure 1), and 12 of them were third-generation cephalosporin-resistant E. coli (

Table 1. HPCIA-resistant E. coli isolates with different ST and resistance genes combinations.

ID Isolates	Sequence Type	Resistance Genes
		3GC *	Fosfomycin	Ciprofloxacin
003-C1	101	blaCTX-M-14	-	-
004-X1	101	blaCTX-M-14	-	-
013-X1	117	blaCTX-M-55	fosL1	qnrB19
014-X1	117	blaCTX-M-55	fosL1	qnrB19
050-X1	48	blaCMY-2	-	qnrS1
054-X1	23	blaCTX-M-55	fosA3	qnrB19
071-X1	23	blaCTX-M-14	-	qnrS1
091-X1	23	blaCTX-M-14	-	-
103-X1	58	blaCTX-M-55	-	-
112-X1	10	blaCTX-M-55	fosA3	qnrB19
122-X1	744	blaCTX-M-55/blaCTX-M-8	fosA3	-
124-X1	88	blaCTX-M-55	-	-

* 3GC: third-generation cephalosporin.

).

4. Discussion

In Argentina, most studies on this topic originate primarily from human sources and, to a lesser extent, from animal production systems. Therefore, comparisons will be made with available regional data. In this study, a significant proportion of HPCIA-resistant E. coli for human health was observed, with a positivity rate of 52.2% in the analysed pork meat samples.

When comparing with the findings of other authors [10,11,12], who analysed isolates from food-producing animals in Argentina (specifically pigs and cattle), the 3GC-R mechanisms differed in frequency; the bla_CTX-M-8 variant gene was the most frequently detected, whereas in our study, bla_CTX-M-55 was the predominant one. Nevertheless, the bla_CMY-2, bla_CTX-M-55, and bla_CTX-M-14 findings in those studies highlight some degree of overlap. The presence of bla_CTX-M-55 may indicate a shift in resistance gene frequency, possibly associated with the genes that confer resistance to fosfomycin, fosA3, or fosL1.

Mounsey et al. [10], who analysed isolates from food-producing animals in Argentina (specifically pigs and cattle), also detected several high-risk clones, such as ST58, ST48, ST101, ST744, and ST117, in swine isolates, consistent with our findings.

In comparison to clinical studies, such as that by Gramundi et al. [13], our clonal profile shows a clear contrast. While they identified ST131 as the most prevalent clone in hospital-associated human isolates, this clone was not detected in our study. This divergence supports the hypothesis that clonal distribution and resistance mechanisms may be strongly influenced by the ecological niche (human, animal, environmental, or food-related) and maybe geographical area. However, the study by Sanz et al. [14], also based on human isolates, reported clones that coincide with our findings, such as ST10 and ST744, although the resistance mechanisms associated with them were different.

The review conducted by Bastida-Caldes et al. [15], which analysed studies from South America between 1990 and 2021, highlights bla_CTX-M-8, bla_CTX-M-2, and bla_CTX-M-1 as the most frequently detected resistance genes in animals in Argentina. In Brazil, bla_CTX-M-2 and bla_CTX-M-8 were predominant in both animal and food samples, while in Uruguay, bla_CTX-M-8 was the most common. These findings differ from our results. The same review identifies ST131 as the most prevalent clone in the region, likely due to the predominance of clinical studies in human populations. Nonetheless, it also reports the presence of ST10, ST117, ST410, and ST744 clones that match those found in our research.

At the regional level, the studies by Soncini et al. in 2022 and 2024 [4,16], which analysed isolates from human sources as well as chicken and pork meat, are also relevant. In both studies, ST131 was the most frequently sequenced type detected clone of epidemiological concern; however, this clone was not identified in our research. Importantly, high-risk clones such as ST10, ST23, ST117, and ST58 were identified in both our study and theirs, all of which were associated with bla_CTX-M-55, the most prevalent resistance gene in our data.

5. Conclusions

This study highlights the importance of food products and the food production chain as reservoirs of high-risk clones and resistance genes of epidemiological relevance to public health. Complementary studies will be necessary to assess the current situation in the region and will help implement the measures needed to address this issue.