Next Article in Journal
Clonal Diversity of Extraintestinal Pathogenic Escherichia coli Strains Isolated from Canine Urinary Tract Infections in Brazil
Previous Article in Journal
Prevalence and Characterization of the Antimicrobial Resistance and Virulence Profiles of Staphylococcus aureus in Ready-to-Eat (Meat, Chicken, and Tuna) Pizzas in Mansoura City, Egypt
Previous Article in Special Issue
Raw Sheep Milk as a Reservoir of Multidrug-Resistant Staphylococcus aureus: Evidence from Traditional Farming Systems in Romania
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Antibiotics
Volume 14
Issue 8
10.3390/antibiotics14080818
antibiotics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Colistin-Resistant Escherichia coli Isolated from Houseflies and Feces of Cattle and Pigs at a Slaughterhouse in Lima, Peru

by
Andrea Carhuallanqui
1,
Lorena Villafana
1,
Rosa Gonzalez-Veliz
2,
José F. Cobo-Díaz
3,
Avelino Álvarez-Ordoñez
3 and
Daphne Doris Ramos-Delgado
1,*
1
Public Health and Environmental Health Laboratory, Universidad Nacional Mayor de San Marcos, Lima 15081, Peru
2
Avian Pathology Laboratory, Universidad Nacional Mayor de San Marcos, Lima 15081, Peru
3
Department of Food Hygiene and Technology and Institute of Food Science and Technology, Universidad de León, 24004 León, Spain
*
Author to whom correspondence should be addressed.
Antibiotics 2025, 14(8), 818; https://doi.org/10.3390/antibiotics14080818
Submission received: 10 June 2025 / Revised: 25 July 2025 / Accepted: 7 August 2025 / Published: 10 August 2025
(This article belongs to the Special Issue Virulence, Antimicrobial Resistance and Biofilm Production in Veterinary, Zoonotic and Food-Related Pathogens)
Browse Figures
Versions Notes

Abstract

Background: Pigs and cattle have been implicated as reservoirs of antimicrobial resistance genes (ARGs) that can spread to humans, and houseflies are considered potential carriers of bacteria with ARGs that could contribute to their spread to the environment, including food, animals, and humans. Methods: In this study, 107, 145, and 127 Escherichia coli strains were isolated from houseflies, pigs, and cattle, respectively, from a slaughterhouse in Lima, Peru. Antimicrobial susceptibility testing was performed using the Kirby–Bauer method, where thirteen antibiotics were used. Strains were also plated on CHROMagar COL-APSE agar, and colistin’s minimum inhibitory concentration (MIC) was determined. Colistin-resistant E. coli strains were subjected to whole genome sequencing. Results: 7.8% (8/107), 1.38% (2/145), and 0.79% (1/127) of E. coli strains isolated from houseflies, pigs, and cattle, respectively, were resistant to colistin (MIC ≥ 4 µg/mL). ARGs associated with resistance to more than 6 different antibiotic classes were identified, including tetracyclines, beta-lactams, fluoroquinolones, nitroimidazoles, trimethoprim and amphenicols. Conclusions: This study suggests that flies could contribute to the dissemination of ARG carrying bacteria and shows the potential risk of animals and meat production systems as reservoirs of ARG carrying bacteria.

1. Introduction

The spread of antibiotic-resistant bacteria has become a multi-sectoral and multi-factorial global problem, causing at least 700,000 deaths per year worldwide, and is estimated to reach ten million deaths by 2050 [1]. The misuse and overuse of antibiotics in animal agriculture, particularly as growth promoters, prophylaxis, and metaphylaxis, has significantly contributed to the emergence and spread of antimicrobial resistance, with implications for human and environmental health [2].
Colistin, or polyxin E, is considered a last-resort antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections in humans [3]. However, it was widely used in animal agriculture for the prevention and treatment of infections caused by Enterobacteriaceae and as a growth promoter in animal feed for human consumption, making its resistance a concern [4,5].
Colistin resistance can be caused by efflux pumps, loss of lipid A, and the decrease in the net negative charge of the bacterial outer membrane due to the addition of cationic groups (L-Ara4N and pEtN) to lipopolysaccharide [6]. Genetic determinants responsible for colistin resistance include chromosomal mutations in the genes mgrB, prmE, pmrAB, phoQ, arnT, or eptA [7,8,9]. The first plasmid-mediated colistin resistance gene (mcr-1) present in Escherichia coli was reported in 2015 [10] in an isolate from China, and nine mcr homologs have been reported since then [11]. Horizontal transfer of the mcr-1 gene has emerged as a significant contributor to the dissemination of colistin resistance among various Gram-negative bacteria [4,12]. The global spread of the mcr-1 gene suggests that the use of colistin in veterinary medicine has likely accelerated the spread of this gene between humans and animals [13]. Ten different variants of the mcr gene have been reported worldwide (mcr-1 to mcr-10) in clinical and environmental settings, representing a significant public health problem [12,14].
The housefly (Musca domestica) is a synanthropic insect with a worldwide distribution found in homes, restaurants, slaughterhouses, and animal production environments [15,16]. Furthermore, it comes into direct contact with contaminated areas, such as landfills and wastewater; therefore, it can be a vehicle for the transport of bacteria to animals and humans [15]. When feeding on contaminated fluids, the fly can pick up bacteria and these bacteria can multiply in their digestive tract [17]. As a result, houseflies can transmit pathogenic bacteria through regurgitation, mechanical translocation, and defecation [18,19]. Studies have shown that houseflies can be potential vectors of antimicrobial-resistant pathogenic Enterobacteriaceae in agricultural and slaughterhouse settings, posing a risk for transmission of resistant bacteria to the environment, animals, and humans [20,21,22,23].
Few studies have been conducted in Peru on antibiotic resistance in enterobacteria isolated from houseflies [19]. This study aims to pheno- and geno-typically characterize colistin-resistant E. coli isolated from houseflies and feces of cattle and pigs at slaughterhouses in Lima. Additionally, it seeks to expand and strengthen knowledge of antimicrobial resistance in Peru.

2. Results

All E. coli strains were confirmed by biochemical testing, and a total of 379 E. coli strains were obtained. A total of 107, 145, and 127 E. coli strains were isolated from houseflies, pigs, and cattle, respectively. Of the E. coli isolates from houseflies, pigs, and cattle, 7.48% (8/107), 1.38% (2/145), and 0.79% (1/127), respectively, were resistant to colistin.

2.1. Antimicrobial Susceptibility Testing

One-hundred-and-seven strains of E. coli were isolated from house flies, and resistance to lincomycin (100%), tetracycline (90.65%), ampicillin (74.77%), amoxicillin (72.90%), and chloramphenicol (69.16%) was reported. (Table 1). One-hundred-and-forty-five strains of E. coli were isolated from pig feces, and resistance to lincomycin (100%), tetracycline (97.24%), chloramphenicol (92.41%), ampicillin (89.66%), and sulfamethoxazole-trimethoprim (77.93%) was reported (Table 1). One-hundred-and-twenty-seven strains of E. coli were isolated from cattle feces, and resistance to lincomycin (100%), tetracycline (81.89%), chloramphenicol (22.05%), amoxicillin (20.47%), and ampicillin (18.90%) was reported (Table 1).
Pig feces showed a significantly higher prevalence of multidrug-resistant E. coli strains (93.73%) than E. coli strains isolated from house flies (71.96%) (p < 0.001). Likewise, flies and pig feces showed a significantly higher prevalence than E. coli strains isolated from cattle (19.68%) (p < 0.001). The multidrug-resistant E. coli strains were resistant to ampicillin, chloramphenicol, sulfamethoxazole-trimethoprim, ciprofloxacin, tetracycline, and gentamicin.

2.2. Colistin Resistance

The 7.48% (8/107), 1.38% (2/145), and 0.79% (1/127) of the E. coli isolates from houseflies, pigs, and cattle, respectively, were resistant to colistin (MIC ≥ 4 µg/mL); and mcr-1 gene was not detected, by PCR, in these isolates. The eleven colistin-resistant E. coli strains were multidrug-resistant (Table 2), and their complete genomes were sequenced, employing the following codes for the genomes labeling: pig (203B and 150B), houseflies (107M, 84M, 110M, 61M, 81M, 95M, 21M, and 65M) and cattle (131V).

2.3. Serotype and Multilocus Sequence Typing (MLST)

Nine E. coli strains were typed considering serogroup O, whereas two E. coli strains isolated from flies were unassignable (non-typeable serogroup O). All 11 strains were assigned to a known serogroup H. Overall, ten different H types and six O types were identified (Table 3). Achtman’s seven-locus MLST scheme was used, and nine different sequence types (STs) were identified, but two E. coli strains (from flies and pigs) could not be assigned to known STs (Table 3; Figure 1).

2.4. Virulence Genes

One E. coli strain isolated from flies had the yuA gene, and one E. coli strain isolated from pigs had the ironN gene, both genes associated with iron acquisition systems. The E. coli strain isolated from bovine feces presented the f17d-G gene (fimbrial-type adhesin). All strains presented the fimH gene (fimbria type 1 adhesin) and the ompA gene (serum resistance) (Table 4). No major virulence determinants associated with E. coli pathotypes, such as hlyA, stx, elt, eae, aggR, ipaH, and hlyA genes, were detected.

2.5. Antimicrobial Resistance Genes and Chromosomal Gene Mutations Associated with Antimicrobial Resistance

ARGs associated with resistance to seven different antibiotic classes were identified: aminoglycosides (aadA3, aadA, aadA2 and aph(3’)-la), tetracyclines (tet(A), tet(M)), beta-lactams (blaTEM-1, blaTEM-176), fluoroquinolones (qnrs1, qnrsB10 and qnrB19), trimethoprim (dfrA8), sulfonamides (sul1 and sul3), and amphenicols (cmlA1). In addition, more than fifty point mutations in intrinsic chromosomal genes associated with antimicrobial resistance were identified (Figure 2).
The 11 E. coli strains did not present mobile colistin resistance genes; however, all genomes contained chromosomal point mutations previously linked to colisin resistance (arnT, eptA, and pmrF).
Fourteen plasmidic contigs with ARGs were identified in four E. coli strains: 107M (1 plasmidic contig), 84M (2), 150B (5), and 203B (6). They harbored tet(A), dfrA12, dfrA1, dfrA8, aadA2, aadA3, blaTEM-1, blaTEM-176, qnrS1, qnrB10, qnrB19, APH(3′)-Ia, sul3, sul1, qacEdelta1, and linG (Figure 3).

2.6. Phylogenetic Analysis

The genomes were grouped into two clusters: 107M (fly), 110M (fly), 150B (pig), and 203B (pig), representing the first cluster, and 65M, 61M, 21M, 84M, 95M, and 81M (flies), and 131V (cattle), representing the second cluster. cgMLST analysis revealed a shared ancestry between the single E. coli genome isolated from cattle (131V) and an E. coli genome isolated from a fly (21M). Furthermore, it revealed a common ancestry between two E. coli genomes isolated from pigs (150B and 203B) and one E. coli genome isolated from a fly (110M) (Figure 4).

3. Discussion

In the present study, a total of 379 strains of E. coli were isolated from domestic flies (107), pigs (145), and cattle (127). 93.73%, 71.96%, and 19.68% of the E. coli strains isolated from pigs, flies, and cattle, respectively, were multidrug-resistant (to ampicillin, chloramphenicol, sulfamethoxazole-trimethoprim, ciprofloxacin, tetracycline, and gentamicin) (Table 1). This study demonstrates that asymptomatic cattle and pigs slaughtered at a slaughterhouse in Lima carry E. coli resistant to various antibiotics. E. coli strains isolated from cattle feces showed significantly lower multidrug resistance than E. coli strains isolated from pigs and flies. The difference between cattle and pigs is due to the type of livestock system and stocking density in both production systems. Cattle are generally raised in an extensive livestock system, while pig farming is an intensive livestock system. Antimicrobials are routinely used to prevent disease in intensive systems, contributing significantly to the emergence of antimicrobial resistance in pigs [24,25]. Furthermore, flies present in the slaughterhouse are in constant contact with animal feces, which can spread resistant bacteria.
International guidelines on the use of antibiotics as growth promoters vary across countries. European countries and the US have banned antibiotics as growth promoters in animal feed [26,27,28]. China also adopted this legislation in 2020, banning 11 antibiotics [29]. Growth promoters based on all antibiotic classes for all animal species have been banned in Chile and Colombia; Argentina only allows mixing certain antibiotics, none of which are critically important for human medicine; Brazil has been phasing out many antibiotic classes; Uruguay prohibits using antibiotics as growth promoters in sheep and cattle, but no legislation was found regarding their use in pigs and chickens [30]. In contrast, Peru does not have legislation prohibiting antibiotics in growth promoters; only antibiotics such as chloramphenicol, nitrofurans, olaquindox, nitroimidazoles, and colistin have been banned.
The Kirby–Bauer method for antimicrobial resistance profiling revealed a higher percentage of resistance to most antibiotics in E. coli strains isolated from pigs compared to those from cattle. Globally, antimicrobial consumption is estimated to be higher in pigs compared to cattle [31,32]. Subtherapeutic antibiotics have been used for many years in food production animals to control the spread of infectious diseases among crowded animals and improve production performance, especially in pig and poultry farming. In pig production, antibiotics can be applied to entire groups by mixing them in feed and water [33].
The resistance profile, analysed using the Kirby–Bauer method, of the E. coli strains isolated from cattle and pig feces showed a tetracycline resistance of 81.89% and 97.24%, respectively. Studies in Latin America also reported high percentages of tetracycline-resistant E. coli. Melgarejo-Touche et al. [34] evaluated antimicrobial resistance in stool samples from beef cattle slaughtered in Asunción (Paraguay) slaughterhouses and reported that 100% of fluoroquinolone-resistant E. coli were resistant to tetracycline. Cabrera González et al. [35] evaluated the resistance of E. coli isolated from the diarrheal feces of newborn calves in the Cajamarca region (Peru) and they found resistance to tetracycline (96.15%), sulfamethoxazole-trimethoprim (51.92%), neomycin (26.92%), and enrofloxacin (9.61%).
The use of tetracyclines as feed additives and drugs for animals intended for human consumption is not forbidden in Peru and it may be the main cause of the high percentage of tetracycline-resistant E. coli strains. The use of tetracyclines in veterinary medicine is common due to several advantages, including low cost, oral administration, and broad spectrum of activity [36]. Genes associated with tetracycline resistance were found in colistin-resistant E. coli strains isolated from pigs (tet(A), tet(M)); point mutations in the genes emrK, emrY, evgA, and evgS were also detected in E. coli strains isolated from pigs and cattle. tet genes generally cause tetracycline resistance and, in the case of Gram-negative bacteria such as E. coli, encode efflux pump systems [36]. tet genes were found in the two E. coli strains isolated from pigs and two E. coli strains isolated from flies (84M and 107M). tet(A) was found on three plasmids in E. coli isolated from houseflies (2) and pigs (1). Therefore, the high frequency may also be due to the horizontal transfer of these genes, as has been reported recently [37].
All E. coli strains isolated from pigs, cattle, and flies were resistant to lincomycin; this may be due to cross-resistance with macrolides and group B streptogramin (MLSB) caused by ribosomal modification by methylases encoded by erm genes [38,39]. The 11 colistin-resistant E. coli strains exhibited point mutations in the erm(A) and erm(B) genes, conferring resistance to lincomycin. This resistance could be due to the use of macrolides as growth promoters in cattle and pigs [40]. On the other hand, most E. coli strains isolated from flies were resistant to ampicillin (74.77%) and tetracycline (90.65%). The percentage of resistance detected could be a consequence of the high consumption of these antimicrobials in pig and beef cattle production [35,40,41]. Other studies also reported that enterobacteria isolated from house flies were frequently resistant to tetracycline and ampicillin [42,43,44].
The Peruvian Ministry of Agriculture and Irrigation (MINAGRI) banned the import, marketing, and manufacture of active ingredients of chloramphenicol, nitrofurans (furazolidone and nitrofurantoin), olaquindox and nitroimidazoles in Peru in 2013 and in 2019 colistin was banned for use in animals [45]. However, despite the ban, colistin-resistant bacteria have been reported in animals for human consumption [41]. Chloramphenicol resistance possibly originated through mutation of the acrA, acrB and tolC genes because the AcrAB efflux system belongs to the nodulation-division transporter (NDT) family and uses the TolC system. Substrates for this efflux system (AcrABTolC) have included chloramphenicol, tetracyclines, fluoroquinolones, and trimethoprim [46,47]. In addition, this study reported resistance to nitrofurantoin, using the Kirby–Bauer method, in E. coli strains isolated from pigs (1.38%) and flies (3.74%). However, in the 11 colistin-resistant E. coli strains, no genes associated with nitrofurantoin resistance were found.
Phylogenetically, eleven colistin-resistant E. coli strains belonged to group A (3/11) and B1 (8/11), being classified as non-pathogenic commensal strains, representing a reduced risk to the environment and public health [48]. This is consistent with other studies that reported that E. coli strains isolated from cattle and pigs most frequently belong to group B1 and/or A [49,50]. The E. coli strains isolated from pigs (150B and 203B) were identified to present the same phylogroup as a strain isolated from M. domestica (110M). Likewise, E. coli strains with the same phylogroup for flies (107M, 21M, 61M, 65M, 81M, 84M, and 95M) as for cattle (131V) were identified.
The eleven isolates sequenced in this study belong to different sequence types, suggesting a diversity in colistin-resistant E. coli strains. The use of media not supplemented with colistin eliminated the selective pressure and limited the recovery of colistin-resistant E. coli. This work revealed a diverse resistome with ARGs that confer resistance to more than 10 antibiotic classes, including chloramphenicol, tetracycline, aminoglycosides, and ciprofloxacin. These results demonstrate the circulation of colistin-resistant E. coli carrying different antibiotic-resistance genes in houseflies and cattle in Lima. This is consistent with projections, which indicate that Latin America is increasing the consumption of antimicrobials by cattle [51].
Plasmids can disseminate antimicrobial resistance genes in Gram-negative bacteria. They generally harbor determinants that confer resistance to different classes of antibiotics simultaneously; in this study, plasmids with resistance genes to tetracyclines, fluoroquinolones, ESBLs, aminoglycosides, lincomycins, trimethoprim, and sulfonamides were found. The following incompatibility plasmids were found: IncFIB (AP001918) (107M, 84M) and IncX1 (150B), and a colicinogenic plasmid in strain 84M (Col(pHAD28)). In addition, the IncX1 plasmid presented the APH genes aph(3′)-Ia and blaTEM-176; plasmid families that include IncX play an important role in the propagation of ESBL genes [52]. Plasmids IncFIB (AP001918) and Col(pHAD28) were also reported in E. coli strains in drinking water and feces of dogs and cattle in rural areas of Cajamarca, Peru [53].
The two genomes of the E. coli strains isolated from pigs presented five (150B) and six plasmidic contigs (203B) with antimicrobial resistance genes. The qacEdelt1 gene was identified in a plasmid of one E. coli strain isolated from pigs (203B), together with ESBL genes and genes of resistance to sulfonamides, lincomycin, fluoroquinolones, and tetracyclines. This indicates possible co-resistance to disinfectants such as quaternary ammonium compounds [54,55]. In addition, there are studies where the qacEdelt1 gene has been considered a genetic marker of class 1 integrons associated with multidrug-resistant phenotypes [56].
There are several antibiotics used in animals for human consumption and classified as critically important antimicrobials for the treatment of people; among them is colistin, which, according to the World Health Organization’s list of critically important antimicrobials for human medicine, is considered among those with the “highest priority” [57]. Only eleven strains of E. coli were phenotypically resistant to colistin, and none presented mcr genes. The eleven E. coli strains presented point mutations in the arnT and eptA genes that can individually or collectively affect the efficiency of the phosphoethanolamine transferase enzyme, altering the structure of the bacterial membrane and reducing the ability of colistin to bind and exert its antimicrobial effect [9].
The most common mechanism of colistin resistance is due to the mutation of chromosomal genes associated with the modification of lipid A of LPS, the main target of colistin, as an adaptive response [58]. Such modifications can be obtained by the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) and phosphoethanolamine (PEtN) to lipid A [59] (Baron et al., 2016). Genes encoding enzymes involved in lipid A synthesis are pmrHFIJFKLM (arnBCADTEF-pmrE). These genes are positively regulated by the two-component systems PhoPQ and PmrAB [9]. Resistance to colistin has been previously linked to mutations in pmrAB and phoPQ (two-component regulatory systems) [7]. The arnBCADTEF operon, which is activated by PmrAB, triggers the synthesis and addition of L-Ara4N to lipid A, reducing the binding affinity to colistin [60]. In addition, eptA, which PmrAB also activates, is involved in the synthesis and addition of PEtN; consequently, L-Ara4N and PEtN are added to LPS, decreasing the negative charge of the outer membrane and reducing the binding affinity to colistin [61].
All 11 colistin-resistant strains presented point mutations in the mdtM gene. MdtM is a monocomponent multidrug efflux protein that belongs to the major facilitator superfamily and interacts with membrane-spanning transporters, such as the AcrABeTolC complex, to form a coordinated supersystem of membrane transporters that confers antimicrobial resistance [62]. The mutation of the mdtM gene in all E. coli strain genomes is likely because its multidrug efflux activity is a co-opted adaptation of its physiological functions; therefore, it persists in the bacterial genome even in the absence of pharmacological selective pressure [63].
Multidrug resistance is most frequently found in pigs, followed by flies, and less frequently in cattle. These E. coli strains can be transmitted to humans through the food chain, posing a public health risk. Animal waste can also spread antimicrobial-resistant bacteria into the environment [64]. Furthermore, phylogenetic analysis showed a possible circulation of colistin-resistant E. coli between animals in the slaughterhouse and houseflies. This study found that colistin-resistant E. coli strains presented plasmids with resistance genes to different antimicrobials; this indicates a significant risk for public health, being able to transmit genes to other Gram-negative bacteria horizontally [65]. The housefly could transmit these bacteria through regurgitation of vomit and defecation. In addition, “bioenhanced transmission” has been reported, where bacteria can multiply at regurgitation sites, mouthparts, and the intestine [18]. From pigs and cattle and through houseflies, these E. coli strains could enter the food chain to contaminate food and other environments. The frequency of antimicrobial resistance in pigs reported in the present study highlights the urgent need to restrict and ensure the prudent use of antimicrobials in Peru.
This study showed that houseflies collected inside the slaughterhouse presented E. coli strains resistant to different antimicrobials, possibly because the flies easily acquired the bacteria when they fed on the manure and waste found in the slaughterhouse [66]. However, they could also have acquired these bacteria from other environments, as they can fly 5–7 km [67,68,69]. Studies have shown the presence of antimicrobial-resistant enterobacteria isolated from houseflies in different environments [18,19,42,70].

4. Materials and Methods

4.1. Flies Sampling and Escherichia coli Isolation

One hundred fifty houseflies were collected using an entomological net from pig and cattle pens at a slaughterhouse in Lima, Peru, from February 2022 to October 2023. Five flies were captured every fifteen days. After collection, the flies were placed in sterile jars and immediately transported at 4 °C to the Public Health and Environmental Health Laboratory of the Faculty of Veterinary Medicine of the Universidad Nacional Mayor de San Marcos (FMV- UNMSM). Each fly was morphologically identified using a stereoscope and following the keys of Greenberg [71]. The main steps of the Pava–Ripoll et al. [72] protocol were followed for the dissection of flies; the flies were sacrificed by freezing them to a temperature of −20 °C and then disinfected with 70% ethanol and washed individually in sterile saline solution.
The digestive tracts of the flies were removed with fine-tipped dissecting forceps and placed individually in 5 mL vials containing peptone water. The digestive tract suspensions were homogenized for three minutes and incubated individually at 37 °C for 18 h. After incubation, a 100 μL aliquot was plated on Petri dishes containing eosin methylene blue agar (EMB) (Merck, Darmstadt, Germany). EMB agar plates were then incubated at 37 °C for 24 h. Two suspected E. coli colonies from each fly were streaked on Tryptone Soy Agar (TSA, Merck, Germany) for confirmation with conventional biochemical tests, such as plating on Triple Sugar Iron Agar (TSI, Merck, Germany), Lysine Iron Agar (LIA, Merck, Germany), Sulfide Indole Mobility (SIM, Merck, Germany), Urea Broth (Merck, Germany) and Simmons Citrate Agar (Merck, Germany), following the procedures described elsewhere [73,74,75]. All confirmed E. coli strains were subjected to antimicrobial susceptibility testing against 13 commonly used human and veterinary medicine antibiotics.

4.2. Collection of Feces from Pigs and Cattle and Isolation of E. coli

In addition to house flies, aseptically collected fecal samples were obtained from cattle (n = 150) and pigs (n = 150) in the same slaughterhouse and time as the flies. Sampling was carried out in the abdominal viscera washing area of the slaughterhouse, making an incision in the intestine approximately 65 cm proximal to the anal sphincter [76]. Approximately 150 g of feces were collected from the distal area of the large intestine using a sterile polyethylene bag. Five samples of each species were taken every fifteen days. The samples were transported at 4 °C immediately to the laboratory for processing.
E. coli enrichments were prepared by diluting 25 g of feces in 225 mL of buffered peptone water (Merck, Germany). Subsequently, each enrichment was homogenized for 1 min and incubated at 42 °C for 18 h [76]. For the isolation of E. coli, 100 µL of peptone water was plated on EMB agar. The EMB plates were incubated at 37 °C for 24 h. For confirmation, two suspected E. coli colonies per sample were plated on TSA agar for confirmation with conventional biochemical tests such as TSI agar, LIA agar, SIM agar, urea broth, and Simmons Citrate agar, and the antimicrobial susceptibility of all confirmed E. coli isolates was tested, as described above for the housefly E. coli isolates.

4.3. Antimicrobial Susceptibility Testing

A total of 107, 145, and 127 E. coli strains were isolated from houseflies, pigs, and cattle, respectively, and were tested for antimicrobial susceptibility. The Kirby–Bauer disk diffusion method was used on cation-adjusted Mueller Hinton agar (Merck, Germany) [77]. The antibiotic disks used were as follows: amoxicillin (20 μg), ampicillin (10 μg), cephalexin (30 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), enrofloxacin (5 μg), gentamicin (10 μg), lincomycin (2 μg), nalidixic acid (30 μg), neomycin (30 μg), nitrofurantoin (300 μg), sulfamethoxazole-trimethoprim (1.25/23.75 μg), and tetracycline (30 μg). Results were interpreted according to CLSI guidelines [77].
Moreover, E. coli strains were grown on CHROMagar COL-APSE agar (CHROMagar, La Plaine St-Denis, France) at 37 °C to test for colistin resistance. In addition, the minimum inhibitory concentration (MIC) of colisitin was determined by broth microdilution using ComASP™ Colistin (Liofilchem, Roseto degli Abruzzi, Italy) and following the manufacturer’s recommendations. Escherichia coli ATCC 25922 and a molecularly characterized E. coli strain CP141, carrying the mcr-1 gene, from the Public Health and Environmental Health Laboratory of FMV-UNMSM were used for quality control of the tests.

4.4. Detection of the mcr-1 Gene

The possible presence of E. coli mcr-1-positive strains was assessed by conventional PCR in all E. coli strains with a colistin MIC > 2 μg/mL. For that, E. coli isolates were grown overnight on TSA agar (Merck, Germany), and genomic DNA was extracted using the GeneJET Genomic DNA Purification kit (Thermo Scientific, Waltham, MA, USA), following the manufacturer’s protocol. A NanoDrop One spectrophotometer (ThermoFisher Scientific, USA) was used to determine DNA purity and concentration. Finally, the DNA was stored at −20 °C until further analysis. The PCR analysis was performed using as primers CLR5-F (5′-CGGTCAGTCCGTTTGTTC-3′) and CLR5-R (5′-CTTGGTCGGTCTGTAGGG-3′), as described by Liu et al. [10].
A final reaction volume of 25 µL was used (23 µL of Master Mix and 2 µL of DNA). The Master Mix consisted of 12.5 µL of DreamTaq Green PCR Master Mix 2× (Thermo Fisher Scientific™, USA), 0.75 µL of Primer Forward (10 µM), 0.75 µL of Primer Reverse (10 µM), and 9 µL of nuclease-free water (Thermo Fisher Scientific™, USA) [78]. Reactions were carried out in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). Initial denaturation was performed at 94 °C for 5 min; 35 cycles, with an amplification cycle at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s; a final elongation at 72 °C for 5 min was added [78].
PCR amplicons were visualized on an UltraPure™ Agarose gel (Thermo Scientific, USA) in 1× UltraPure™ 10× TBE buffer (Thermo Scientific, USA), prestained with UltraPure™ Ethidium Bromide (Invitrogen, Carlsbad, CA, USA), under UV transillumination. Electrophoresis was performed at 100 volts for 62 min.

4.5. Genome Characterization

Whole genome sequencing was performed on all E. coli isolates with a colistin MIC > 2 μg/mL. It was performed by Illumina 150 paired-end sequencing on a NovaSeq 600 platform (Illumina, Inc., San Diego, CA, USA). Raw reads were quality filtered by Trimmomatic v0.36 [79] and filtered reads were assembled with SPAdes v3.13.0 [80], using default parameters (BioProject PRJNA1274168).
The MLST tool (https://github.com/tseemann/mlst, accessed on 20 December 2024) and SerotypeFinder v.2.0 from the Center for Genomic Epidemiology (CGE) (http://www.genomicepidemiology.org, accessed on 20 December 2024) were used to determine the sequence types and serotypes, respectively, of the E. coli genomes [81]. The EzClermont tool (https://nickp60.github.io/EzClermont/, accessed on 3 January 2025) was used to predict phylogroups [82]. All tools were employed using default settings.
Virulence genes were annotated using the CGE VirulenceFinder database with ABRICATE v.1.0.1 tool (https://github.com/tseemann/abricate, accessed on 10 January 2025), while CGE ResFinder v4.6.0 (http://www.genomicepidemiology.org, accessed on 20 August 2024) and the CARD database (https://card.mcmaster.ca/, accessed on 20 August 2024) were used to detect antimicrobial resistance genes, considering 80% coverage and identity cutoffs. Finally, plasmid types were predicted using the Plasmidfinder v2.1 web server [83] (https://cge.food.dtu.dk/services/PlasmidFinder/, accessed on 21 September 2024).

4.6. cgMLST Hierarchical Clustering Analysis

Genome Profiler v1.0.1 (https://github.com/jizhang-nz/fast-GeP, accessed on 30 July 2024) was used for cgMLST analysis using E. coli str. K-12 substr. MG1655 (NCBI: txid511145) as reference genome [84,85]. Genes were concatenated into a FASTA file using the Ruby script extract_concat_cgMLST_genes.rb (https://github.com/JoseCoboDiaz/concat_cgMLST_genes, accessed on 20 July 2024) for phylogenetic tree construction. This file was used for alignment, and a phylogenetic tree was constructed with MAFFT version 7 [86] using the neighbor-joining method and default parameters for alignment [87], the Jukes-Cantor substitution model and a 1000 bootstrap resampling. The ‘ape’ and the ‘ggtree’ R-packages were used to read the Newick files and plot the phylogenetic tree, respectively. The phylogenetic tree was edited to its final version using InkScape software (https://inkscape.org/).

4.7. Statistical Analysis

The data were analyzed using Stata version 14.0 (StataCorp, College Station, TX, USA). Descriptive statistics were used to determine the frequencies of resistant E. coli strains for each species (flies, cattle, and pigs). The chi-square test was used to determine the significance of the differences observed between the three species and the multidrug-resistant E. coli strains.

5. Conclusions

This study observed that the prevalence of multidrug resistance to antimicrobials in E. coli strains isolated from cattle feces in a slaughterhouse was low compared to pig and housefly feces.
The genomes of multidrug-resistant and phenotypically colistin-resistant E. coli strains isolated from cattle, pigs, and flies displayed chromosomal point mutations linked to colistin resistance. Furthermore, colistin-resistant E. coli strains were shown to have plasmid contigs containing a variety of resistance genes.
Our findings show that cattle, pigs, and flies can be reservoirs of colistin-resistant E. coli strains, and the house fly is a potential disseminator of antimicrobial-resistant genes from the slaughterhouse to other environments. Furthermore, ongoing surveillance using a One Health approach is urgently needed to monitor the emergence and spread of antimicrobial-resistance genes.

Author Contributions

Conceptualization, D.D.R.-D. and A.C.; methodology, A.C., L.V., R.G.-V., J.F.C.-D. and A.Á.-O.; validation, D.D.R.-D., A.C., J.F.C.-D., R.G.-V. and A.Á.-O.; investigation, A.C., L.V., R.G.-V., J.F.C.-D., and A.Á.-O.; data curation, A.C., L.V., J.F.C.-D., and A.Á.-O.; writing—original draft preparation, A.C., J.F.C.-D. and A.Á.-O.; writing—review and editing, A.C., J.F.C.-D., A.Á.-O. and D.D.R.-D.; funding acquisition, A.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was subsidized by Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica (CONCYTEC) and Programa Nacional de Investigación Científica y Estudios Avanzados (PROCIENCIA) in the framework of the “Concurso E073-2023-01- Tesis de Pregrado y Postgrado en Ciencia, Tecnología e Innovación Tecnológica” contest, according to contract (PE501081775-2022).

Institutional Review Board Statement

Ethical review and approval were waived for this study because the flies were collected at the slaughterhouse, and fecal samples from pigs and cattle were collected in the abdominal viscera washing area of the slaughterhouse with the owner’s permission.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. O’Neill, J. Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations. The Review on Antimicrobial Resistance, Chaired by Jim O’Neill. 2014, pp. 1–20. Available online: https://amr-review.org/sites/default/files/AMR%20Review%20Paper%20-%20Tackling%20a%20crisis%20for%20the%20health%20and%20wealth%20of%20nations_1.pdf (accessed on 3 January 2025).
  2. Stehling, E.G.; Calero-Cáceres, W.; Makita, K.; Furlan, J.P.R. Editorial: Livestock and its role in the emergence, spread, and evolution of antimicrobial resistance: Animal-to-human or animal-to-environment transmission. Front. Vet. Sci. 2023, 10, 1270955. [Google Scholar] [CrossRef]
  3. Rhouma, M.; Madec, J.Y.; Laxminarayan, R. Colistin: From the shadows to a One Health approach for addressing antimicrobial resistance. Int. J. Antimicrob. Agents 2023, 61, 106713. [Google Scholar] [CrossRef]
  4. Poirel, L.; Madec, J.Y.; Lupo, A.; Schink, A.K.; Kieffer, N.; Nordmann, P.; Schwarz, S. Antimicrobial Resistance in Escherichia coli. Microbiol. Spectr. 2018, 6, 10-1128/microbiolspec.arba-0026-2017. [Google Scholar] [CrossRef]
  5. Shen, Y.; Zhang, R.; Schwarz, S.; Wu, C.; Shen, J.; Walsh, T.R.; Wang, Y. Farm animals and aquaculture: Significant reservoirs of mobile colistin resistance genes. Environ. Microbiol. 2020, 22, 2469–2484. [Google Scholar] [CrossRef]
  6. Moffatt, J.H.; Harper, M.; Boyce, J.D. Mechanisms of Polymyxin Resistance. Adv. Exp. Med. Biol. 2019, 1145, 55–71. [Google Scholar] [CrossRef]
  7. Poirel, L.; Jayol, A.; Nordmann, P. Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes. Clin. Microbiol. Rev. 2017, 30, 557–596. [Google Scholar] [CrossRef]
  8. Kim, S.; Woo, J.H.; Kim, N.; Kim, M.H.; Kim, S.Y.; Son, J.H.; Moon, D.C.; Lim, S.K.; Shin, M.; Lee, J.C. Characterization of Chromosome-Mediated Colistin Resistance in Escherichia coli Isolates from Livestock in Korea. Infect. Drug Resist. 2019, 12, 3291–3299. [Google Scholar] [CrossRef] [PubMed]
  9. Hamel, M.; Rolain, J.M.; Baron, S.A. The History of Colistin Resistance Mechanisms in Bacteria: Progress and Challenges. Microorganisms 2021, 9, 442. [Google Scholar] [CrossRef] [PubMed]
  10. Liu, Y.Y.; Wang, Y.; Walsh, T.R.; Yi, L.X.; Zhang, R.; Spencer, J.; Doi, Y.; Tian, G.; Dong, B.; Huang, X.; et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: A microbiological and molecular biological study. Lancet Infect. Dis. 2016, 16, 161–168. [Google Scholar] [CrossRef] [PubMed]
  11. Hussein, N.H.; Al-Kadmy, I.M.S.; Taha, B.M.; Hussein, J.D. Mobilized colistin resistance (mcr) genes from 1 to 10: A comprehensive review. Mol. Biol. Rep. 2021, 48, 2897–2907. [Google Scholar] [CrossRef]
  12. Liu, J.H.; Liu, Y.Y.; Shen, Y.B.; Yang, J.; Walsh, T.R.; Wang, Y.; Shen, J. Plasmid-mediated colistin-resistance genes: Mcr. Trends Microbiol. 2024, 32, 365–378. [Google Scholar] [CrossRef]
  13. El-Sayed Ahmed, M.A.E.; Zhong, L.L.; Shen, C.; Yang, Y.; Doi, Y.; Tian, G.B. Colistin and its role in the Era of antibiotic resistance: An extended review (2000–2019). Emerg. Microbes Infect. 2020, 9, 868–885. [Google Scholar] [CrossRef]
  14. Mondal, A.H.; Khare, K.; Saxena, P.; Debnath, P.; Mukhopadhyay, K.; Yadav, D. A Review on Colistin Resistance: An Antibiotic of Last Resort. Microorganisms 2024, 12, 772. [Google Scholar] [CrossRef] [PubMed]
  15. Förster, M.; Klimpel, S.; Sievert, K. The house fly (Musca domestica) as a potential vector of metazoan parasites caught in a pig-pen in Germany. Vet. Parasitol. 2009, 160, 163–167. [Google Scholar] [CrossRef] [PubMed]
  16. Nazari, M.; Mehrabi, T.; Hosseini, S.M.; Alikhani, M.Y. Bacterial Contamination of Adult House Flies (Musca domestica) and Sensitivity of these Bacteria to Various Antibiotics, Captured from Hamadan City, Iran. J. Clin. Diagn. Res. 2017, 11, DC04–DC07. [Google Scholar] [CrossRef] [PubMed]
  17. Onwugamba, F.C.; Mellmann, A.; Nwaugo, V.O.; Süselbeck, B.; Schaumburg, F. Antimicrobial resistant and enteropathogenic bacteria in ‘filth flies’: A cross-sectional study from Nigeria. Sci. Rep. 2020, 10, 16990. [Google Scholar] [CrossRef]
  18. Onwugamba, F.C.; Fitzgerald, J.R.; Rochon, K.; Guardabassi, L.; Alabi, A.; Kühne, S.; Grobusch, M.P.; Schaumburg, F. The role of ‘filth flies’ in the spread of antimicrobial resistance. Travel. Med. Infect. Dis. 2018, 22, 8–17. [Google Scholar] [CrossRef]
  19. Alarcón-Calle, M.A.; Osorio-Guevara, V.L.; Salas-Asencios, R.; Yareta, J.; Marcos-Carbajal, P.; Rodrigo-Rojas, M.E. Carbapenems and colistin resistance genes isolated in Musca domestica from a garbage dump near a hospital in Lima. Genes resistentes a carbapenémicos y colistina aislados en Musca domestica proveniente de un basural cercano a un hospital de Lima. Rev. Peru. Med. Exp. Salud Publica 2024, 41, 164–170. [Google Scholar] [CrossRef]
  20. Ranjbar, R.; Izadi, M.; Hafshejani, T.T.; Khamesipour, F. Molecular detection and antimicrobial resistance of Klebsiella pneumoniae from house flies (Musca domestica) in kitchens, farms, hospitals and slaughterhouses. J. Infect. Public Health 2016, 9, 499–505. [Google Scholar] [CrossRef]
  21. Ommi, D.; Hemmatinezhad, B.; Hafshejani, T.T.; Khamesipour, F. Incidencia y resistencia antimicrobiana de Campylobacter y Salmonella de moscas domésticas (Musca domestica) en cocinas, granjas, hospitales y mataderos. Proc. Natl. Acad. Sci. India Sect. B Biol. Sci. 2017, 87, 1285–1291. [Google Scholar] [CrossRef]
  22. Yun, B.; Jang, Y.J.; Kim, Y.R.; Kim, H.Y.; Kim, W.I.; Han, S.; Kim, S.R.; Ryu, J.G.; Kim, H.J. Virulence profile and antimicrobial resistance of Escherichia coli from flies captured from agricultural environment. J. Food Hyg. Saf. 2017, 32, 147–153. [Google Scholar] [CrossRef]
  23. Odetoyin, B.; Adeola, B.; Olaniran, O. Frequency and Antimicrobial Resistance Patterns of Bacterial Species Isolated from the Body Surface of the Housefly (Musca domestica) in Akure, Ondo State, Nigeria. J. Arthropod Borne Dis. 2020, 14, 88–96. [Google Scholar] [CrossRef]
  24. Van Boeckel, T.P.; Pires, J.; Silvester, R.; Zhao, C.; Song, J.; Criscuolo, N.G.; Gilbert, M.; Bonhoeffer, S.; Laxminarayan, R. Global trends in antimicrobial resistance in animals in low- and middle-income countries. Science 2019, 365, eaaw1944. [Google Scholar] [CrossRef]
  25. Stevenson, P. Links between industrial livestock production, disease including zoonoses and antimicrobial resistance. Anim. Res. One Health 2023, 1, 137–144. [Google Scholar] [CrossRef]
  26. Castanon, J.I. History of the use of antibiotic as growth promoters in European poultry feeds. Poult. Sci. 2007, 86, 2466–2471. [Google Scholar] [CrossRef] [PubMed]
  27. Huyghebaert, G.; Ducatelle, R.; Van Immerseel, F. An update on alternatives to antimicrobial growth promoters for broilers. Vet. J. 2011, 187, 182–188. [Google Scholar] [CrossRef] [PubMed]
  28. Low, C.X.; Tan, L.T.; Ab Mutalib, N.S.; Pusparajah, P.; Goh, B.H.; Chan, K.G.; Letchumanan, V.; Lee, L.H. Unveiling the Impact of Antibiotics and Alternative Methods for Animal Husbandry: A Review. Antibiotics 2021, 10, 578. [Google Scholar] [CrossRef] [PubMed]
  29. Wen, R.; Li, C.; Zhao, M.; Wang, H.; Tang, Y. Withdrawal of antibiotic growth promoters in China and its impact on the foodborne pathogen Campylobacter coli of swine origin. Front. Microbiol. 2022, 13, 1004725. [Google Scholar] [CrossRef]
  30. Da Silva, R.A.; Arenas, N.E.; Luiza, V.L.; Bermudez, J.A.Z.; Clarke, S.E. Regulations on the Use of Antibiotics in Livestock Production in South America: A Comparative Literature Analysis. Antibiotics 2023, 12, 1303. [Google Scholar] [CrossRef] [PubMed]
  31. Van Boeckel, T.P.; Brower, C.; Gilbert, M.; Grenfell, B.T.; Levin, S.A.; Robinson, T.P.; Teillant, A.; Laxminarayan, R. Global trends in antimicrobial use in food animals. Proc. Natl. Acad. Sci. USA 2015, 112, 5649–5654. [Google Scholar] [CrossRef]
  32. Lekagul, A.; Tangcharoensathien, V.; Mills, A.; Rushton, J.; Yeung, S. How antibiotics are used in pig farming: A mixed-methods study of pig farmers, feed mills and veterinarians in Thailand. BMJ Glob. Health 2020, 5, e001918. [Google Scholar] [CrossRef] [PubMed]
  33. Filippitzi, M.; Callens, B.; Pardon, B.; Persoons, D.; Dewulf, J. Antimicrobial use in pigs, broilers and veal calves in Belgium. Vlaams Diergeneeskd. Tijdschrift. 2014, 83, 215–224. [Google Scholar] [CrossRef]
  34. Melgarejo-Touchet, N.; Busignani, S.; Dunjo, P.; Brítez, M.; Weiler, N.; Orrego, V.; Alonzo, M.; Martínez-Mora, M. Resistencia antimicrobiana en Escherichia coli de muestras cecales de bovinos para carne faenados en frigoríficos de la zona del arroyo Mburicao, Asunción-Paraguay. Año 2021. Mem. Inst. Investig. Cienc. Salud 2022, 20, 51–59. [Google Scholar] [CrossRef]
  35. Cabrera González, M.A.; Vásquez Pérez, H.V.; Quilcate-Pairazamán, C.; Bazán-Arce, J.; Cueva-Rodríguez, M. Evaluación de resistencia a antibióticos en muestras de heces de terneros con diarrea en la región Cajamarca, Perú. Rev. Mex. Cienc. Pecu. 2023, 14, 782–795. [Google Scholar] [CrossRef]
  36. Chopra, I.; Roberts, M. Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev. 2001, 65, 232–260. [Google Scholar] [CrossRef]
  37. Tokuda, M.; Shintani, M. Microbial evolution through horizontal gene transfer by mobile genetic elements. Microb. Biotechnol. 2024, 17, e14408. [Google Scholar] [CrossRef]
  38. Clarebout, G.; Nativelle, E.; Leclercq, R. Unusual Inducible Cross Resistance to Macrolides, Lincosamides, and Streptogramins B by Methylase Production in Clinical Isolates of Staphylococcus aureus. Microb. Drug Resist. 2001, 7, 317–322. [Google Scholar] [CrossRef]
  39. Svetlov, M.S.; Syroegin, E.A.; Aleksandrova, E.V.; Atkinson, G.C.; Gregory, S.T.; Mankin, A.S.; Polikanov, Y.S. Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance. Nat. Chem. Biol. 2021, 17, 412–420. [Google Scholar] [CrossRef] [PubMed]
  40. Ríos, C.A.; Morales-Cauti, S.; Vilca, L.M.; Carhuallanqui, A.; Ramos, D. Determinación del perfil de resistencia antibiótica de Salmonella enterica aislada de cerdos faenados en un matadero de Lima, Perú. Rev. Investig. Vet. Perú 2019, 30, 438–445. [Google Scholar] [CrossRef]
  41. Carhuaricra, D.; Duran Gonzales, C.G.; Rodríguez Cueva, C.L.; Ignacion León, Y.; Silvestre Espejo, T.; Marcelo Monge, G.; Rosadio Alcántara, R.H.; Lincopan, N.; Espinoza, L.L.; Maturrano Hernández, L. Occurrence and Genomic Characterization of mcr-1-Harboring Escherichia coli Isolates from Chicken and Pig Farms in Lima, Peru. Antibiotics 2022, 11, 1781. [Google Scholar] [CrossRef]
  42. Bertelloni, F.; Bresciani, F.; Cagnoli, G.; Scotti, B.; Lazzerini, L.; Marcucci, M.; Colombani, G.; Bilei, S.; Bossù, T.; De Marchis, M.L.; et al. House Flies (Musca domestica) from Swine and Poultry Farms Carrying Antimicrobial Resistant Enterobacteriaceae and Salmonella. Vet. Sci. 2023, 10, 118. [Google Scholar] [CrossRef]
  43. Cervelin, V.; Fongaro, G.; Pastore, J.B.; Engel, F.; Reimers, M.A.; Viancelli, A. Enterobacteria associated with houseflies (Musca domestica) as an infection risk indicator in swine production farms. Acta Trop. 2018, 185, 13–17. [Google Scholar] [CrossRef]
  44. Akter, S.; Sabuj, A.A.M.; Haque, Z.F.; Kafi, M.A.; Rahman, M.T.; Saha, S. Detection of antibiotic-resistant bacteria and their resistance genes from houseflies. Vet. World. 2020, 13, 266–274. [Google Scholar] [CrossRef]
  45. Iglesias-Osores, S. Uso de colistina en el sector pecuario: Necesidad de una prohibición global. Acta Med. Peru. 2020, 37, 114–115. [Google Scholar] [CrossRef]
  46. Ma, Q.P.; Su, L.; Liu, J.W.; Yao, M.X.; Yuan, G.Y. Study on the association between drug resistance and gene mutations of the active efflux pump acrAB tolC gene and its regulatory genes. Mol. Med. Rep. 2018, 17, 8228–8236. [Google Scholar] [CrossRef]
  47. Yaqoob, M.; Wang, L.P.; Kashif, J.; Memon, J.; Umar, S.; Iqbal, M.F.; Fiaz, M.; Lu, C.P. Genetic characterization of phenicol-resistant Escherichia coli and role of wild-type repressor/regulator gene (acrR) on phenicol resistance. Folia Microbiol. 2018, 63, 443–449. [Google Scholar] [CrossRef]
  48. Chakraborty, A.; Saralaya, V.; Adhikari, P.; Shenoy, S.; Baliga, S.; Hegde, A. Characterization of Escherichia coli Phylogenetic Groups Associated with Extraintestinal Infections in South Indian Population. Ann. Med. Health Sci. Res. 2015, 5, 241–246. [Google Scholar] [CrossRef] [PubMed]
  49. Ewers, C.; de Jong, A.; Prenger-Berninghoff, E.; El Garch, F.; Leidner, U.; Tiwari, S.K.; Semmler, T. Genomic Diversity and Virulence Potential of ESBL- and AmpC-β-Lactamase-Producing Escherichia coli Strains From Healthy Food Animals Across Europe. Front. Microbiol. 2021, 12, 626774. [Google Scholar] [CrossRef] [PubMed]
  50. Messele, Y.E.; Trott, D.J.; Hasoon, M.F.; Veltman, T.; McMeniman, J.P.; Kidd, S.P.; Djordjevic, S.P.; Petrovski, K.R.; Low, W.Y. Phylogenetic Analysis of Escherichia coli Isolated from Australian Feedlot Cattle in Comparison to Pig Faecal and Poultry/Human Extraintestinal Isolates. Antibiotics 2023, 12, 895. [Google Scholar] [CrossRef]
  51. Mulchandani, R.; Wang, Y.; Gilbert, M.; Van Boeckel, T.P. Global trends in antimicrobial use in food-producing animals: 2020 to 2030. PLoS Glob. Public Health 2023, 3, e0001305. [Google Scholar] [CrossRef] [PubMed]
  52. Carattoli, A. Resistance Plasmid Families in Enterobacteriaceae. Antimicrob. Agents Chemother. 2009, 53, 2227–2238. [Google Scholar] [CrossRef]
  53. Medina-Pizzali, M.L.; Venkatesh, A.; Riveros, M.; Cuicapuza, D.; Salmon-Mulanovich, G.; Mäusezahl, D.; Hartinger, S.M. Whole-Genome Characterisation of ESBL-Producing E. coli Isolated from Drinking Water and Dog Faeces from Rural Andean Households in Peru. Antibiotics 2022, 11, 692. [Google Scholar] [CrossRef]
  54. Liu, X.; Gong, L.; Liu, E.; Li, C.; Wang, Y.; Liang, J. Characterization of the Disinfectant Resistance Genes qacEΔ1 and cepA in Carbapenem-Resistant Klebsiella pneumoniae Isolates. Am. J. Trop. Med. Hyg. 2023, 110, 136–141. [Google Scholar] [CrossRef]
  55. Kovalchuk, S.N.; Arkhipova, A.L.; Bondar, S.V.; Konanov, D.N.; Krivonos, D.V.; Chulkova, P.S.; Ageevets, V.A.; Fedorova, L.S.; Ilina, E.N. A TaqMan real-time PCR assay for detection of qacEΔ1 gene in Gram-negative bacteria. J. Microbiol. Methods 2024, 227, 107054. [Google Scholar] [CrossRef]
  56. Chen, S.; Fu, J.; Zhao, K.; Yang, S.; Li, C.; Penttinen, P.; Ao, X.; Liu, A.; Hu, K.; Li, J.; et al. Class 1 integron carrying qacEΔ1 gene confers resistance to disinfectant and antibiotics in Salmonella. Int. J. Food Microbiol. 2023, 404, 110319. [Google Scholar] [CrossRef]
  57. World Health Organisation WHO. List of Critically Important Antimicrobials for Human Medicine. Available online: https://www.who.int/publications/i/item/9789241515528 (accessed on 19 April 2024).
  58. Olaitan, A.O.; Morand, S.; Rolain, J.M. Mechanisms of polymyxin resistance: Acquired and intrinsic resistance in bacteria. Front. Microbiol. 2014, 5, 643. [Google Scholar] [CrossRef]
  59. Baron, S.; Hadjadj, L.; Rolain, J.M.; Olaitan, A.O. Molecular mechanisms of polymyxin resistance: Knowns and unknowns. Int. J. Antimicrob. Agents 2016, 48, 583–591. [Google Scholar] [CrossRef]
  60. Jeannot, K.; Bolard, A.; Plésiat, P. Resistance to polymyxins in Gram-negative organisms. Int. J. Antimicrob. Agents 2017, 49, 526–535. [Google Scholar] [CrossRef] [PubMed]
  61. Choi, Y.; Lee, J.Y.; Lee, H.; Park, M.; Kang, K.; Lim, S.K.; Shin, D.; Ko, K.S. Comparison of Fitness Cost and Virulence in Chromosome- and Plasmid-Mediated Colistin-Resistant Escherichia coli. Front. Microbiol. 2020, 11, 798. [Google Scholar] [CrossRef] [PubMed]
  62. Law, C.J.; Alegre, K.O. Clamping down on drugs: The Escherichia coli multidrug efflux protein MdtM. Res. Microbiol. 2018, 169, 461–467. [Google Scholar] [CrossRef] [PubMed]
  63. Holdsworth, S.R.; Law, C.J. Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in Escherichia coli. BMC Microbiol. 2013, 13, 113. [Google Scholar] [CrossRef]
  64. Song, H.J.; Kim, S.J.; Moon, D.C.; Mechesso, A.F.; Choi, J.H.; Kang, H.Y.; Boby, N.; Yoon, S.S.; Lim, S.K. Antimicrobial Resistance in Escherichia coli Isolates from Healthy Food Animals in South Korea, 2010–2020. Microorganisms 2022, 10, 524. [Google Scholar] [CrossRef]
  65. Sobur, M.A.; Ievy, S.; Haque, Z.F.; Nahar, A.; Zaman, S.B.; Rahman, M.T. Emergence of colistin-resistant Escherichia coli in poultry, house flies, and pond water in Mymensingh, Bangladesh. J. Adv. Vet. Anim. Res. 2019, 6, 50–53. [Google Scholar] [CrossRef] [PubMed]
  66. Gwenzi, W.; Chaukura, N.; Muisa-Zikali, N.; Teta, C.; Musvuugwa, T.; Rzymski, P.; Abia, A.L.K. Insects, Rodents, and Pets as Reservoirs, Vectors, and Sentinels of Antimicrobial Resistance. Antibiotics 2021, 10, 68. [Google Scholar] [CrossRef] [PubMed]
  67. Skovgård, H.; Nachman, G. Population dynamics of stable flies Stomoxys calcitrans (Diptera: Muscidae) at an organic dairy farm in Denmark based on mark-recapture with destructive sub-sampling. Environ. Entomol. 2012, 41, 20–29. [Google Scholar] [CrossRef]
  68. Farag, T.H.; Faruque, A.S.; Wu, Y.; Das, S.K.; Hossain, A.; Ahmed, S.; Ahmed, D.; Nasrin, D.; Kotloff, K.L.; Panchilangam, S.; et al. Housefly population density correlates with shigellosis among children in Mirzapur, Bangladesh: A time series analysis. PLoS Negl. Trop. Dis. 2013, 7, e2280. [Google Scholar] [CrossRef] [PubMed]
  69. Stelder, J.J.; Kjær, L.J.; Jensen, L.B.; Boklund, A.E.; Denwood, M.; Carlsen, M.; Bødker, R. Livestock-associated MRSA survival on house flies (Musca domestica) and stable flies (Stomoxys calcitrans) after removal from a Danish pig farm. Sci. Rep. 2021, 11, 3527. [Google Scholar] [CrossRef]
  70. Wetzker, W.; Pfeifer, Y.; Wolke, S.; Haselbeck, A.; Leistner, R.; Kola, A.; Gastmeier, P.; Salm, F. Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli Isolated from Flies in the Urban Center of Berlin, Germany. Int. J. Environ. Res. Public Health 2019, 16, 1530. [Google Scholar] [CrossRef]
  71. Greenberg, B. Flies and Disease, 1st ed.; Princeton University Press: Princeton, NJ, USA, 1971; Volume 1, pp. 856–863. [Google Scholar]
  72. Pava-Ripoll, M.; Pearson, R.E.; Miller, A.K.; Ziobro, G.C. Detection of foodborne bacterial pathogens from individual filth flies. J. Vis. Exp. 2015, 96, e52372. [Google Scholar] [CrossRef]
  73. Alves, T.D.S.; Lara, G.H.B.; Maluta, R.P.; Ribeiro, M.G.; Leite, D.D.S. Carrier flies of multidrug-resistant Escherichia coli as potential dissemination agent in dairy farm environment. Sci. Total Environ. 2018, 633, 1345–1351. [Google Scholar] [CrossRef]
  74. Ewing, W.H. Edwards and Ewing’s Identification of Enterobacteriaceae, 4th ed.; Elsevier: New York, NY, USA, 1986; pp. 181–201. [Google Scholar]
  75. Feng, P.; Weagant, S.D.; Grant, M.A.; Burkhardt, W.; Shellfish, M.; Water, B. BAM: Enumeration of Escherichia coli and the Coliform Bacteria. In Bacteriological Analytical Manual; US Food & Drug Administration: Silver Spring, MD, USA, 2002; Volume 13, pp. 1–13. [Google Scholar]
  76. Barlow, R.; McMillan, K.; Mellor, G.; Duffy, L.; Jordan, D.; Abraham, R.; O’Dea, M.; Sahibzada, S.; Abraham, S. Phenotypic and Genotypic Assessment of Antimicrobial Resistance in Escherichia coli from Australian Cattle Populations at Slaughter. J. Food Prot. 2022, 85, 563–570. [Google Scholar] [CrossRef]
  77. M100; Performance Standards for Antimicrobial Susceptibility Testing, 31st ed. Clinical and Laboratory Standards Institute (CLSI): Wayne, PA, USA, 2021.
  78. Institute of Infectious Diseases. National Administration of Laboratories and Health Institutes “Dr. Carlos, G. Malbrán”. PCR Protocol for the Detection of the Mcr-1 Gene in Gram-Negative Bacilli Isolates. Available online: https://antimicrobianos.com.ar/ATB/wp-content/uploads/2021/04/Detecci%c3%b3n-mcr-1-v1.pdf (accessed on 20 April 2024).
  79. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef]
  80. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef] [PubMed]
  81. Joensen, K.G.; Tetzschner, A.M.; Iguchi, A.; Aarestrup, F.M.; Scheutz, F. Rapid and Easy In Silico Serotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data. J. Clin. Microbiol. 2015, 53, 2410–2426. [Google Scholar] [CrossRef]
  82. Waters, N.R.; Abram, F.; Brennan, F.; Holmes, A.; Pritchard, L. Easy phylotyping of Escherichia coli via the EzClermont web app and command-line tool. Access Microbiol. 2020, 2, acmi000143. [Google Scholar] [CrossRef] [PubMed]
  83. Carattoli, A.; Zankari, E.; García-Fernández, A.; Voldby Larsen, M.; Lund, O.; Villa, L.; Møller Aarestrup, F.; Hasman, H. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob. Agents Chemother. 2024, 58, 3895–3903. [Google Scholar] [CrossRef] [PubMed]
  84. Hayashi, K.; Morooka, N.; Yamamoto, Y.; Fujita, K.; Isono, K.; Choi, S.; Ohtsubo, E.; Baba, T.; Wanner, B.L.; Mori, H.; et al. Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Mol. Syst. Biol. 2006, 2, 2006-0007. [Google Scholar] [CrossRef]
  85. Zhang, J.; Xiong, Y.; Rogers, L.; Carter, G.P.; French, N. Genome-by-genome approach for fast bacterial genealogical relationship evaluation. Bioinformatics 2018, 34, 3025–3027. [Google Scholar] [CrossRef]
  86. Katoh, K.; Rozewicki, J.; Yamada, K.D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2018, 20, 1160–1166. [Google Scholar] [CrossRef]
  87. Bryant, D.; Moulton, V. Neighbor-Net: An agglomerative method for the construction of phylogenetic networks. Mol. Biol. Evol. 2004, 21, 255–265. [Google Scholar] [CrossRef]
Antibiotics 14 00818 g001
Figure 1. GrapeTree based on MLST scheme of 11 E. coli genes according to species of origin and sequence type [1,2,8].
Figure 1. GrapeTree based on MLST scheme of 11 E. coli genes according to species of origin and sequence type [1,2,8].
Antibiotics 14 00818 g001
Antibiotics 14 00818 g002
Figure 2. Heatmap (presence/absence) of acquired antimicrobial resistance genes (ARGs) and chromosomal gene mutations associated with antimicrobial resistance (CGMAR) of E. coli isolated from flies, pigs, and cattle. Red squares indicate presence and blue squares absence of ARG/CGMAR.
Figure 2. Heatmap (presence/absence) of acquired antimicrobial resistance genes (ARGs) and chromosomal gene mutations associated with antimicrobial resistance (CGMAR) of E. coli isolated from flies, pigs, and cattle. Red squares indicate presence and blue squares absence of ARG/CGMAR.
Antibiotics 14 00818 g002
Antibiotics 14 00818 g003
Figure 3. Plasmidic contigs with resistance genes from colistin-resistant E. coli strains isolated from pigs and houseflies (107M, 84M: housefly; 150B, 203B: pig).
Figure 3. Plasmidic contigs with resistance genes from colistin-resistant E. coli strains isolated from pigs and houseflies (107M, 84M: housefly; 150B, 203B: pig).
Antibiotics 14 00818 g003
Antibiotics 14 00818 g004
Figure 4. cgMLST phylogenetic analysis of genomic sequences obtained from E. coli strains isolated from flies, cattle and pigs.
Figure 4. cgMLST phylogenetic analysis of genomic sequences obtained from E. coli strains isolated from flies, cattle and pigs.
Antibiotics 14 00818 g004
Table 1. Antimicrobial resistance prevalence of Escherichia coli strains using the Kirby–Bauer method.
Table 1. Antimicrobial resistance prevalence of Escherichia coli strains using the Kirby–Bauer method.
AntibioticsCattle (%)
n = 150		Pig (%)
n = 150		Fly (%)
n = 150
Lincomycin100100100
Enrofloxacin11.8138.6240.19
Tetracycline81.8997.2490.65
Neomycin8.6653.149.53
Ampicillin18.989.6674.77
Amoxicillin20.4789.6672.9
Chloramphenicol22.0592.4169.16
Nalidixic Acid23.6270.3463.55
Sulfatrimethoprim16.5477.9363.55
Colistin0.791.387.48
Ciprofloxacin4.7231.7239.25
Nitrofurantoin01.383.74
Cephalexin4.726.930.84
Gentamicin6.327.5932.71
Multidrug resistance19.6893.7371.96
Table 2. Antimicrobial resistance profile of colistin-resistant Escherichia coli strains.
Table 2. Antimicrobial resistance profile of colistin-resistant Escherichia coli strains.
AntibioticsCattlePigFly
131V150B203B21M61M65M81M84M95M107M110M
LincomycinRRRRRRRRRRR
EnrofloxacinRRRRSRSSSRS
TetracyclineRRRRRRRRRRR
NeomycinRRRRRRRRRRR
AmpicillinSRRRSRRRSRS
AmoxicillinSRRSSSSRSRS
ChloramphenicolRRRRRRRRRRR
Nalidixic AcidRRRRRRRRRRR
SulfatrimethoprimSRRRSSSRSRS
CiprofloxacinRRRRRRRRRRR
NitrofurantoinSSSSSSSSSSS
CephalexinSRRSSSSSSSS
GentamicinRRRRRRRRRRR
R: resistant; S: susceptible.
Table 3. Summary of the characterization of 11 colistin-resistant E. coli isolates (sequence, serotype and phylogroup).
Table 3. Summary of the characterization of 11 colistin-resistant E. coli isolates (sequence, serotype and phylogroup).
StrainSpeciesSTSerotypePhylogroupClonal Complex
107Mfly-O13 H29B1-
110MflyST 1408O13 H30A-
131VcattleST 937O43 H2B1-
150BpigST 1115O102 H40A-
203Bpig-O102 H20A-
21MflyST 201O3 H19B1ST469 Cplx
61MflyST 1252O13 H11B1-
65MflyST 6333O123 H14B1-
81MflyST 278H7B1ST278 Cplx
84MflyST 5487H25B1-
95MflyST 351O18 H7B1-
Table 4. Virulence genes of 11 colistin-resistant E. coli strains isolated from flies, cattle and pigs.
Table 4. Virulence genes of 11 colistin-resistant E. coli strains isolated from flies, cattle and pigs.
GenesIsolates
FlyCattlePig
107M110M21M61M65M81M84M95M131V150B203B
algW+++++++++++
aslA+++++++++++
cheA−−+++++++−−
cheD+++++++++++
cheY+++++++++++
csgB+++++++++++
csgD+++++++++++
csgE+++++++++++
csgF+++++++++++
csgG+++++++++++
entA+++++++++++
entB+++++++++++
entC+++++++++++
entD+++++++++++
entE+++++++++++
entF+++++++++++
entS+++++++++++
espL1+++++++++++
espL4+++++++++++
espR1+++++++++++
espX1+++++++++++
espX4+++++++++++
espX5+++++++++++
espR4++−−−−−−−−−
espX6+−−−−−−−−−−
espY2+−−−−−−−−−−
espY4+−−−−−−−−−−
fdeC+++++++++++
fepA+++++++++++
fepB+++++++++++
fepC+++++++++++
fepD+++++++++++
fepG+++++++++++
f17d-A−−−−−−−−+−−
f17d-C−−−−−−−−+−−
f17d-D−−−−−−−−+−−
f17d-G−−−−−−−−+−−
fes+++++++++++
fimA+++++++++++
fimB+++++++++++
fimC+++++++++++
fimD+++++++++++
fimE+++++++++++
fimF+++++++++++
fimG+++++++++++
fimH+++++++++++
fimI+++++++++++
flgD+−+−++−++−−
flgF+−−−−−−−−−−
flgE+++++++++++
flgG+++++++++++
flgH+++++++++++
flgM+++++++++++
flgJ++−+−−+−−++
flhA+++++++++++
fliG+++++++++++
fliI+++++++++++
fliM+++++++++++
fliN+++++++++++
fliP+++++++++++
fliC+−−+−−+−+++
gmd+−−−−−−−−−−
gspC−−+++++−+++
gspD−−+++++−+++
gspE−−+++++−+++
gspF−−+++++−+++
gspG+++++++++++
gspH−−+++++++++
gspI−−+++++++++
gspJ+++++++++++
gspK+++++++++++
gspL+++++++++++
gspM+++++++++++
gtrA++−+−−−−−−−
gtrB++−+−−−−−−−
gtrII++−−−−−−−−−
katB++−++++++++
ompA+++++++++++
sfaF+−−−−−−−−−−
pefC−−+−−−−−−−−
pefD−−+−−−−−−−−
iroB−−−−−−+−−−−
iroC−−−−−−+−−−−
iroD−−−−−−+−−−−
iroE−−−−−−+−−−−
iroN−−−−−−+−−−−
yagV/ecpE+++++++++++
yagW/ecpD+++++++++++
yagX/ecpC+++++++++++
yagY/ecpB+++++++++++
yagZ/ecpA+++++++++++
ykgK/ecpR+++++++++++
irp1−−−+−−−−−−−
irp2−−−+−−−−−−−
fyuA−−−+−−−−−−−
(+): presence; (−): absence.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Carhuallanqui, A.; Villafana, L.; Gonzalez-Veliz, R.; Cobo-Díaz, J.F.; Álvarez-Ordoñez, A.; Ramos-Delgado, D.D. Colistin-Resistant Escherichia coli Isolated from Houseflies and Feces of Cattle and Pigs at a Slaughterhouse in Lima, Peru. Antibiotics 2025, 14, 818. https://doi.org/10.3390/antibiotics14080818

AMA Style

Carhuallanqui A, Villafana L, Gonzalez-Veliz R, Cobo-Díaz JF, Álvarez-Ordoñez A, Ramos-Delgado DD. Colistin-Resistant Escherichia coli Isolated from Houseflies and Feces of Cattle and Pigs at a Slaughterhouse in Lima, Peru. Antibiotics. 2025; 14(8):818. https://doi.org/10.3390/antibiotics14080818

Chicago/Turabian Style

Carhuallanqui, Andrea, Lorena Villafana, Rosa Gonzalez-Veliz, José F. Cobo-Díaz, Avelino Álvarez-Ordoñez, and Daphne Doris Ramos-Delgado. 2025. "Colistin-Resistant Escherichia coli Isolated from Houseflies and Feces of Cattle and Pigs at a Slaughterhouse in Lima, Peru" Antibiotics 14, no. 8: 818. https://doi.org/10.3390/antibiotics14080818

APA Style

Carhuallanqui, A., Villafana, L., Gonzalez-Veliz, R., Cobo-Díaz, J. F., Álvarez-Ordoñez, A., & Ramos-Delgado, D. D. (2025). Colistin-Resistant Escherichia coli Isolated from Houseflies and Feces of Cattle and Pigs at a Slaughterhouse in Lima, Peru. Antibiotics, 14(8), 818. https://doi.org/10.3390/antibiotics14080818

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop