Research Article

Detection and Antimicrobial Susceptibility of Escherichia coli in Retail Chicken Breast Meat in General Santos City, Philippines

Alexis N. Miole1,2, Gabrielle Beatrix B. Francisco3, Chrizyll Joy G. Paulo1,4, Harlequin S. Tamayo1,5, Mahalea U. Sabilla1,6, Michael M. Sebastian1,5, Gibbs Ada Rose B. Cawan1,7, Keiven Mark B. Ampode1,8,9*

1Graduate School, Sultan Kudarat State University, Tacurong City 9800, Philippines; 2Department of Animal Science, College of Agriculture, Mindanao State University - General Santos, General Santos City 9500, Philippines; 3Office of the Vice Chancellor for Research, Extension, and Innovation, Mindanao State University - General Santos, General Santos City 9500, Philippines; 4Agricultural Service Unit, Office of the Municipal Mayor, Local Government of Datu, Abdullah Sangki, Maguindanao del Sur 9609, Philippines; 5Crops Development Section, Field Operation Division, Office of the Provincial Agriculturist, Provincial Government of South Cotabato, City of Koronadal 9506, Philippines; 6Agricultural Engineering and Infrastructure Section, Agricultural Research and Support Services Division, Office of the Provincial Agriculturist, Provincial Government of South Cotabato, City of Koronadal 9506, Philippines; 7Department of Animal Science, College of Agriculture, New Hope School of Agriculture and Fisheries, Polomolok, South Cotabato 9504, Philippines; 8College of Agriculture, Sultan Kudarat State University - Lutayan Campus, Lutayan, Sultan Kudarat, 9803, Philippines; 9College of Agriculture, Forestry and Environmental Sciences, Western Philippines University, Palawan, 5302, Philippines.

Alexis N. Miole and Gabrielle Beatrix B. Francisco contributed equally and share first authorship to this work.

Received | December 22, 2025; Accepted | February 08, 2026; Published | March 08, 2026

*Correspondence | Keiven Mark B. Ampode, College of Agriculture, Sultan Kudarat State University, Lutayan Campus, Tacurong City 9800, Philippines; Email: [email protected]

Citation | Miole AN, Francisco GBB, Paulo CJG, Tamayo HS, Sabilla MU, Sebastian MM, Cawan GARB, Ampode KMB (2026). Detection and antimicrobial susceptibility of Escherichia coli in retail chicken breast meat in general Santos City, Philippines. Adv. Anim. Vet. Sci., 14(3):577-582.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/14.3.577.582

ISSN (Online) | 2307-8316

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

In the Philippines, chicken is a primary source of animal protein due to its affordability, low fat content, and minimal religious or cultural restrictions. The shift in consumer preference from pork to chicken has been further driven by supply shortages and rising pork prices associated with recurrent African Swine Fever outbreaks (Kaye, 2019). As a result, the poultry industry has become one of the country’s fastest-growing livestock sectors (Espino and Bellotindos, 2020).

Antibiotics are extensively used in poultry production for disease prevention and treatment. However, their inappropriate and excessive use has driven the emergence and spread of antimicrobial resistance (AMR), representing a major global public health concern within the One Health framework (Abreu et al., 2023; Pineda-Cortel et al., 2024). In 2019, antimicrobial-resistant bacterial infections were associated with an estimated 4.95 million deaths worldwide, of which approximately 1.27 million were directly attributable to resistant pathogens (Tang et al., 2023). In this context, there is a possibility that resistant bacteria in retail poultry could indirectly contribute to the problem.

Escherichia coli (E. coli) is a commensal bacterium commonly found in the intestinal tract of chickens (Koutsianos et al., 2021). During slaughtering and processing, intestinal contents may contaminate carcasses, potentially transferring E. coli to raw poultry meat. Consumption of improperly handled or undercooked poultry products contaminated with E. coli can therefore transmit these bacteria to humans (Wardhana et al., 2021). Pathogenic strains of E. coli are associated with a range of illnesses, including hemorrhagic enteritis, dysentery, meningitis, pneumonia, and urinary tract infections (Astuti et al., 2021). E. coli is also a significant cause of foodborne infections and outbreaks. Among its pathogenic variants, enterohemorrhagic E. coli (EHEC) has emerged as a major public health threat, accounting for an estimated 9,000 illnesses and 70 deaths annually in the United States alone (Amalia et al., 2020).

Despite the public health implications, data on AMR in E. coli from raw chicken meat sold in public markets in General Santos City remain scarce. Thus, this preliminary and descriptive study aimed to isolate and identify E. coli from raw chicken breast samples obtained from selected public markets in the city and to determine the antimicrobial resistance profiles of the isolates using the disk diffusion method. Specifically, the study evaluated resistance against amoxicillin, doxycycline, and norfloxacin by measuring zones of inhibition and characterizing resistance patterns among the isolates.

Materials and Methods

Meat sample collection

Raw chicken breast meat was collected from vendors in different public markets in General Santos City. Public markets included in this study were selected based on the following criteria: (1) the market has high foot traffic with a high number of customers; (2) the market is in proximity and accessible to at least three highly populated barangays; and (3) the market has at least five (5) raw chicken meat vendors. Details of the vendors and the public markets were not disclosed to protect vendor privacy. This limits the traceability of results and potential follow-up by regulatory agencies

Samples were collected on three separate days. The first was a “lean” day, defined as a day with a relatively lower number of customers as compared to other days of the week; another sampling day represented a “peak” day which is defined as the busiest day of the week due to having the most number of customers as compared to other days of the week; and another sampling day was chosen randomly, excluding the other identified sampling days. Sampling across “lean” and “peak” days was conducted to increase temporal coverage and not intended to assess differences in storage time or market-day effects.

After selecting the marketplace and day of the week based on the parameters of this study, raw chicken breast samples were randomly collected from vendors. Five vendors per marketplace were randomly selected by lot. One chicken breast was purchased from each vendor and pooled into a single sterile polyethylene bag. This method was used to maximize vendor coverage within each market. The study was limited only to nine samples in total.

All samples were coded by market (M1, M2, M3) and collection day (D1, D2, D3). The market and day codes were merged to create an isolate code, such as M1D1, M2D2, M3D3. Samples were stored in a freezer prior to transport to PhilExport Quality Control Laboratory, Fish Port Complex, General Santos City, where the samples were analyzed for the presence of E. coli. The isolation, purification, and identification followed the methods of the US Food and Drug Administration Bacteriological Analytical Manual (US FDA BAM) (Feng et al., 2020). Upon receipt, all E. coli isolates from the PhilExport Quality Control Laboratory were streaked onto Eosin Methylene Blue Agar (EMBA), and microscopy was performed to further confirm the identification of E. coli.

Antibiotic susceptibility test (Kirby-Bauer disk diffusion assay)

All E. coli isolates obtained from the samples were tested for antibiotic susceptibility. Three antibiotics representing different antimicrobial classes, β-lactams, tetracyclines, and fluoroquinolones were used in this study. The antibiotics were selected based on: (1) their frequency of use in the Philippine poultry industry (Barroga et al., 2020); (2) their availability as listed in the Philippine Veterinary Drug Directory (2024); and (3) the availability of corresponding resistance breakpoints in the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2023). The CLSI M07 guidelines (CLSI, 2024) were used as the reference for selecting the antibiotics and their corresponding disk concentrations. The antibiotics tested were amoxicillin (10 µg), doxycycline (30 µg), and norfloxacin (10 µg).

All E. coli isolates were streaked onto sterile nutrient agar (NA) plates and incubated for 18–24 hours at 37 °C (Liaqat et al., 2022). After incubation, morphologically similar colonies were selected and transferred into sterile tubes containing 10 mL of normal saline solution (NSS) (Wiegand et al., 2008). The turbidity of the suspension was adjusted to match a 0.5 McFarland standard by visual comparison using a Wickerham card. Bacterial colonies were added as necessary to adjust the turbidity to match the standard (Hudzicki, 2009; Wiegand et al., 2008).

Subsequently, a sterile cotton swab was immersed in the adjusted suspension and used to streak the entire surface of a Mueller–Hinton Agar (MHA) plate. Streaking was performed three times, rotating the plate approximately 60° between streaks to ensure uniform bacterial distribution. The plates were then left to dry at room temperature for 5 minutes (Hudzicki, 2009). The antibiotic discs were aseptically placed equidistantly on the inoculated MHA plates in accordance with CLSI M07 standards (CLSI, 2024). Three antibiotic discs and one blank disc, which served as a negative control, were placed on each plate. The plates were then inverted and incubated at 37 °C for 24 hours. Three replicates per antibiotic were prepared for each of three independent trials. Following incubation, antimicrobial activity was assessed by measuring the zones of inhibition (ZOI) surrounding each disc. The diameters of the inhibition zones were measured, recorded, and analyzed.

Data analysis

Data analysis was purely descriptive. No statistical analysis was performed due to the exploratory nature of the study and limited sample size. This pilot study was intended to establish feasibility of comprehensive, large-scale surveillance studies. The CLSI M100 guidelines (CLSI, 2023) were used as the reference for assessing the antimicrobial resistance profiles of the isolates. Isolates were classified as susceptible when zone diameters were equal to or greater than the susceptible breakpoint, intermediate when zone diameters fell within the intermediate range, and resistant when zone diameters were equal to or less than the resistant breakpoint. The breakpoint values for the antibiotics evaluated in this study are presented in Table 1.

 

Table 1: Reference table for the interpretation of zone diameter breakpoints from CLSI M100.

Antibiotic	Zone diameter breakpoint, nearest whole mm
	Susceptible	Intermediate	Resistant
Amoxicillin, 10 ug	≥17	14-16	≤13
Doxycycline, 30 ug	≥14	11-13	≤10
Norfloxacin, 10 ug	≥17	13-16	≤12

 

(CLSI, 2023).

 

RESULTS AND DISCUSSION

Sample collection

A total of nine samples were collected from different markets on different days. Of these, five samples tested positive for the presence of E. coli. The positive isolates were M1D2, M2D1, M2D3, M3D1, and M3D3. Summary is presented in Table 2.

 

Table 2: Detection of Escherichia coli (E. coli) in chicken breast samples.

Market (M)	Day (D)	Sample Code	E. coli detected
1	1	M1D1	Negative
1	2	M1D2	Positive
1	3	M1D3	Negative
2	1	M2D1	Positive
2	2	M2D2	Negative
2	3	M2D3	Positive
3	1	M3D1	Positive
3	2	M3D2	Negative
3	3	M3D3	Positive

 

Isolation and identification of bacteria

All five isolates tested positive for Indole production and Methyl Red tests, and negative for Voges–Proskauer and Citrate utilization tests (Table 3). Figure 1 shows the morphological characteristics of the isolates. When grown on Eosin Methylene Blue Agar (EMBA), all isolates produced green metallic sheen colonies with a black center (Figure 1A), while on Nutrient Agar (NA), all isolates displayed similar morphological characteristics (Figure 1B), except for isolate M3D1, which had an undulate margin. Gram staining revealed that all isolates retained a pink coloration, confirming that they are Gram-negative. Under 1000× magnification, all isolates were rod-shaped, with cell lengths varying among isolates (Figure 1C).

 

Table 3: Biochemical and phenotypic characteristics of Escherichia coli isolate.

Isolate

Biochemical properties

Grown in eosin-methylene blue agar

Grown in nutrient agar

Microscopy, 1000x magnification

Indole

Methyl Red

Voges- prosk- auer

Citrate

Color

Margin

Elevation

Texture

Color

Margin

Elevation

Texture

Gram stain

Cell shape

M1D2

+

+

-

-

GMS+BC

Entire

Raised

Smooth

Opaque

Entire

Convex

Slimy

Negative

Rods

M2D1

+

+

-

-

GMS+BC

Entire

Raised

Smooth

Opaque

Entire

Convex

Slimy

Negative

Short Rods

M2D3

+

+

-

-

GMS+BC

Entire

Raised

Smooth

Opaque

Entire

Convex

Slimy

Negative

Rods

M3D1

+

+

-

-

GMS+BC

Entire

Raised

Smooth

Opaque

Undulate

Convex

Slimy

Negative

Rods

M3D3

+

+

-

-

GMS+BC

Entire

Raised

Smooth

Opaque

Entire

Convex

Slimy

Negative

Short Rods

 

(+) denotes a positive reaction; (-) denotes a negative reaction; GMS - Green Metallic Sheen; BC - Black Center.

 

E. coli belongs to the Enterobacteriaceae family within the order Enterobacteriales. The cells are straight, cylindrical rods, occurring singly or in pairs, Gram-negative, and aerobic to facultatively anaerobic with a fermentative metabolism. Biochemically, E. coli is positive for indole production and negative for citrate utilization as a carbon source (MacWilliams, 2009; Whitman et al., 2012). Assessment of glucose fermentation end products revealed that all isolates tested positive in the Methyl Red test and negative in the Voges–Proskauer test, consistent with the typical metabolic profile of the genus Escherichia, which produces lactic and acetic acids as end products (McDevitt, 2009). These biochemical characteristics correspond with the observed morphology and reactions of all isolated E. coli strains.

Antibiotic susceptibility test (Kirby-Bauer disk diffusion assay)

All five isolates exhibited resistance to the tested antibiotics: amoxicillin (AMX), doxycycline (DOX), and norfloxacin (NOR) (Table 4). E. coli M3D1 was resistant only to AMX, while E. coli M2D1 was resistant to AMX and NOR. Both E. coli M2D3 and E. coli M1D2 showed resistance to AMX and DOX, whereas E. coli M3D3 was resistant to all three antibiotics (AMX, DOX, and NOR). Figure 2A shows the plate setup. Figure 2B-C show representative results with zones of inhibition.

 

Table 4: Antibiotic resistance profile of Escherichia coli isolates.

Isolate Code	Profile	No. of Antibiotics
M3D1	AMX	1
M2D1	AMX – NOR	2
M2D3	AMX – DOX	2
M1D2	AMX – DOX	2
M3D3	AMX – DOX – NOR	3

 

AMX, Amoxicillin; DOX, Doxycycline; NOR, Norfloxacin

 

E. coli M3D3 is classified as multidrug resistant (MDR), exhibiting resistance to three different classes of antibiotics: Beta-lactam (AMX), Tetracycline (DOX), and

 

 

 

Fluoroquinolone (NOR) (Al-Mustapha et al., 2022). In a larger study by Al-Mustapha et al. (2022), 73 out of 181 chicken broiler cloaca samples tested positive for E. coli, of which 82% were MDR and 50.6% were extensively drug resistant (XDR), defined as resistance to more than five classes of antibiotics. Similarly, Ranabhat et al. (2024) reported E. coli in chicken broiler meat samples consisting of a mixture of breast, liver, and thigh tissues. Out of 105 samples, 61 tested positive for E. coli, with 29.5% classified as MDR.

Among the antibiotics tested in this study, amoxicillin (AMX) was the least effective, with all five isolates (5/5) resistant. For doxycycline (DOX), three of the five isolates (3/5) were resistant, and two out of five (2/5) were susceptible. Norfloxacin (NOR) exhibited two out of five (2/5) resistant, two out of five (2/5) intermediate, and one out of five (1/5) susceptible (Table 5). Rafiq et al. (2024) reported that E. coli isolates from poultry feces were resistant to amoxicillin, with 94.1% classified as multidrug resistant (MDR). Similarly, Gharaibeh et al. (2024) found that 360 of 385 broiler chicken cloaca swab samples tested positive for Avian Pathogenic E. coli (APEC), with 100% resistant to tetracycline and 99.2% resistant to amoxicillin. Larger studies involving cloacal and internal organ isolates were cited to provide a broader context on the occurrence of AMR in the poultry industry.

 

Table 5: Antimicrobial susceptibility of Escherichia coli isolates.

Antibiotic	Susceptibility classification of Escherichia coli isolates based on CLSI M100 guidelines
	Susceptible, isolate count (n/5)	Intermediate, isolate count (n/5)	Resistant, isolate count (n/5)
Amoxicillin, 10 µg	-	-	5/5
Doxycycline, 30 µg	2/5	-	3/5
Norfloxacin, 10 µg	1/5	2/5	2/5

 

(CLSI, 2023); A dash (-) indicates that no isolates were classified in that susceptibility category

 

Conclusion and Recommendation

The preliminary and descriptive findings of this study indicate the presence of multidrug-resistant E. coli in the examined samples of raw chicken breast meat. These findings highlight the urgent need for comprehensive, large-scale surveillance, in collaboration with local regulatory agencies to determine the true prevalence and associated risk factors of antibiotic-resistant E. coli within the Philippine poultry production and distribution chain. Future studies should incorporate appropriate statistical reporting. As a general preventative measure, appropriate food safety practices, including thorough washing, proper cooking, and safe handling of poultry products, are recommended to reduce the risk of foodborne illness and cross-contamination

Acknowledgement

The authors would like to express their sincere gratitude to Dr. Edward Alexander Leyson, Dr. Bernard Bulawan, Dr. Rex Calub, Dr. Remedios Flamiano, Dr. Jane Samejon, Dr. Edward Lapong, Dr. Josh Elisha Octura, Dr. Guillermo Pantuhan, Kathlene Mae Dante, Chenny Rose Rodriguez, Dann Roger Hortal, Tereza Famular for their invaluable guidance and support throughout this study. We also extend our appreciation to the PhilExport Quality Control Laboratory for providing the facilities and technical assistance necessary for the completion of this research.

Novelty Statement

This study provides novel insights into the antimicrobial resistance profiles of E. coli in raw chicken breast sold in public markets in General Santos City, an area with limited published data on foodborne pathogens. Unlike previous research focusing on broiler cloaca or mixed meat samples, this study specifically targets raw chicken breast intended for human consumption, providing direct relevance to food safety and public health. Furthermore, it characterizes the isolates’ resistance against multiple antibiotic classes, identifying the presence of multidrug-resistant strains. The findings not only reveal potential public health risks associated with poultry consumption but also the need for consumer education and regulatory monitoring in the Philippines, thereby contributing to local and global efforts to combat antimicrobial resistance.

Author’s Contribution

ANM and GBF: Contributed to conceptualization, laboratory works, analysis of the data, and manuscript writing. CJGP, HST, MUS, MMS, and GARBC: Assisted in laboratory works. KMBA: Contributed to conceptualization, supervised the study, reviewed protocols, and revised the manuscript.

Generative AI and AI-assisted technology statement

The authors declare that AI-assisted tools (ChatGPT and Grammarly) were used solely to improve English language quality and grammar. All outputs were reviewed by the authors. No AI tools were used for data analysis or interpretation.

Conflict of interest

The authors have declared no conflict of interest.

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