E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668
ORIGINAL RESEARCH ARTICLE
Yakubu Aliyu1*, Justina Adegba2, Fatima Abdullahi Hassan3, Maryam Abdulkarim Ladidi4, Abdullahi Muhammad Shamsuddeen5, and Nusaibah Ahmed Musa6
1,2,3,4,5,6Department of Applied Biology/Microbiology, School of Applied Sciences, Federal Polytechnic, Nasarawa, Nigeria.
This study investigates the antibiotic resistance patterns of beta-lactamase-producing Escherichia coli in Kindirmo in Nasarawa town, Nasarawa State, Nigeria. Over five months, 169 samples were collected from four sampling points. Escherichia coli was isolated using the streak-plating method and identified through biochemical tests. All isolates tested positive for beta-lactamase production. The agar-disc diffusion technique assessed the beta-lactamase-producing E. coli strains' susceptibility to 10 antibiotics. Out of 169 samples, 10 were contaminated with E. coli, resulting in a prevalence of 5.92%. The isolates showed complete resistance to six antibiotics, while 70% and 50% demonstrated susceptibility to ofloxacin, pefloxacin, and gentamicin, respectively. Six antibiotic-resistance phenotypes were identified among the E. coli isolates. The presence of beta-lactamase-producing E. coli in Kindirmo suggests that consuming the product in the area poses a risk to consumers. Authorities should enforce basic hygiene requirements during production and sales to safeguard public health.
Keywords: Antibiotic Resistance, Beta-lactamase, E. coli, Kindirmo, Nasarawa State, Nigeria
Traditional foods, such as milk and its derivatives, constitute vital sources of essential nutrients like high-quality proteins, carbohydrates, fats, B vitamins (B2 and B12), and minerals (iodine, potassium, and phosphorus) (Lucey, 2015). Additionally, milk contains dissolved gases, predominantly carbon dioxide, nitrogen, and oxygen (Lucey, 2015). While milk and its products offer significant nutritional benefits, they can also harbour harmful microorganisms like Salmonella, Escherichia coli, Listeria, Brucella, and Mycobacterium species, posing considerable health risks, especially to vulnerable populations such as individuals with compromised immune systems, older people, pregnant women, and children (FDA, 2020).
Escherichia coli, a notable Gram-negative bacterium, is linked to hospital and community-acquired infections (Wu et al., 2013). The rise of beta-lactamase-producing bacteria, including E. coli, is a significant concern due to the widespread use of cephalosporins. Initially observed in clinical settings, these bacteria are increasingly identified in food-producing animals, contributing to food safety issues with broader environmental ramifications (Agersøn and Aarestrup, 2013; Gandra et al., 2014). Reports suggest that food-producing animals are potential reservoirs for beta-lactamase-producing bacterial pathogens, facilitating their community spread (Veenemans et al., 2014; Dohmen et al., 2015). The escalation of resistance in beta-lactamase-producing bacteria to multiple antibiotics has led to heightened usage of carbapenems (Dahm et al., 2015), which, in turn, has spurred the emergence of carbapenemase-producing bacteria (Liu et al., 2018). Carbapenemase enzymes produced by these bacteria can hydrolyze all beta-lactam antibiotics, posing significant challenges in managing infectious diseases (Badri et al., 2018).
Food safety concerns are particularly complex in many developing countries, characterized by numerous informal markets where a substantial portion of food is sold, making enforcing food safety regulations difficult and often avoided. In Nigeria, hygiene levels and food safety practices among milk and milk product vendors are frequently inadequate. Many rural, suburban, and urban areas lack essential infrastructure and food safety education, leading to compromised sanitary measures, food contamination, and spreading pathogens harboring antibiotic resistance determinants such as beta-lactamase-producing bacteria. The increasing prevalence of beta-lactamase-producing bacteria in communities significantly contributes to the overall burden of antimicrobial resistance (van den Bunt et al., 2017). To our knowledge, no published articles exist on the occurrence and antibiotic resistance patterns of beta-lactamase-producing E. coli strains in milk and milk products from Nasarawa town. In the light of these considerations, this study aimed to determine the antibiotic resistance patterns of beta-lactamase-producing E. coli strains from Kindirmo in Nasarawa town, Nasarawa State, Nigeria.
The research was conducted in Nasarawa town, Nasarawa State, Northern Nigeria. Nasarawa covers an area of approximately 570.4 square kilometers, with a population of 189,835, according to the 2006 census (NBS, 2009). This town is about 10.5 kilometers from Abuja, the Federal Capital Territory, 37 kilometers from Keffi, and 165 kilometres from Lafia, the Nasarawa state capital. The geographical coordinates of the study location are around 8°21'58" N of the equator and 7°5'58" E of the Greenwich Meridian (NBS, 2009).
Sample size
The sample size was determined using the 12.6% prevalence of Escherichia coli in milk, as reported by Reuben et al. (2013). The sample size was determined by using the equation described by Naing et al. (2006):
n = Z2 P (1-P)
d2
Where n= is the sample size;
P is the prevalence from a previous study= 12.6% = 0.126;
Z is the standard normal distribution at 95% confidence interval = 1.96;
d is the absolute desired precision at 5%= 0.05
Therefore, n = (1.96)2 × 0.126× (1-0.126)
(0.05)2
= 0.4231
0.0025
n = 169 samples
Traditionally pasteurised milk (Kindirmo) samples were collected from various locations: Tammah, Gunki, Federal Polytechnic, Nasarawa campus, and Nasarawa main market. Purposive sampling was used to collect the samples based on the concentration of vendors at the different sampling points. The milk samples were collected in sterile, screw-capped containers and transported in an ice-packed cooler to the Microbiology Laboratory of the Department of Applied Biology/Microbiology, Federal Polytechnic, Nasarawa, for analysis.
A 10 ml aliquot of each milk sample was mixed with 90 ml of buffered peptone water and incubated at 37°C for 24 hours under aerobic conditions. After incubation, a loopful of the enriched culture was streaked on Eosin-methylene blue (EMB) agar plates and incubated for another 24 hours at 37°C. Dark-red colonies with a green metallic sheen were considered presumptive E. coli colonies. These presumptive colonies were then sub-cultured onto freshly prepared EMB plates to obtain pure colonies. Purified colonies were streaked onto nutrient agar slants and stored at refrigeration temperature for further identification and characterisation (Cheesbrough, 2010).
The presumptive E. coli isolates were identified using Gram reaction and conventional biochemical tests: indole, methyl-red-Voges-Proskauer, and citrate utilisation tests. Presumptive E. coli colonies were inoculated into nutrient broth and incubated at 37°C for 24 hours. Thereafter, 3 drops of Kovac's reagent were added and shaken gently. Positive reactions were indicated by the development of a red colour in the reagent layer above the broth within 1 minute. In negative reactions, the indole reagent retained its yellow colour. Five (5ml) of methyl red - Voges Proskauer (MRVP) broth was inoculated with presumptive E. coli colonies and incubated at 37°C for 48-72 hours. After this incubation period, 1 ml of the broth was transferred to small serological tubes. To this small quantity, 3 drops of methyl red were added. The development of red colouration on adding the indicator signified a positive result, while a yellow colour showed a negative result. To the rest of the broth in the original tube, 15 drops of 5% - naphthol in ethanol were added, followed by 5 drops of 40% potassium hydroxide. These were mixed by shaking. The caps were loosened, and the bottles were placed in a slanting position. The development of a red colour starting from the liquid-air interface within 1 hour indicated VP positive tests. No colour development occurred in VP negative tests. Simmon's citrate agar slants were prepared in Bijou bottles in accordance with the manufacturer's instructions, and positive colonies obtained from the presumptive tests above were inoculated onto the slants and incubated at 37°C for 24-48 hours with the Bijou bottles loosely capped. A deep blue colour's development indicates a citrate-positive result (Cheesbrough, 2010).
The iodometric method was used to assess the beta-lactamase production in the isolates. This test involves preparing a starch indicator by dissolving starch powder in boiling water and an iodine reagent by mixing potassium iodide and iodine in distilled water. A benzylpenicillin solution in phosphate buffer saline is also prepared and sterilised (Bush and Jacoby, 2010).
To perform the test, a loopful of an overnight-grown beta-lactamase-producing E. coli strain is added to the penicillin G solution and incubated at 37°C for 30 minutes. Two drops of starch solution and one drop of iodine reagent are added to the mixture. Colour changes from dark blue to colourless within 10 minutes, indicating a positive beta-lactamase production test (Bush and Jacoby, 2010).
This method helps identify the presence of beta-lactamase-producing E. coli strains, which play a significant role in antibiotic resistance development, especially to beta-lactam antibiotics.
To prepare the concentration of the suspension for both the test bacterium and the standard strain (E. coli ATCC 25922 obtained from the National Veterinary Research Institute, Vom, Nigeria), a 0.5 scale of McFarland's standard (1.5 × 108 cells/mL) in 0.8% normal saline was used. This was carried out as follows: purified beta-lactamase-producing E. coli colonies were picked using a sterile wire loop. A suspension was prepared by mixing the colonies with normal saline in small tubes. The density of the prepared suspension was then compared with that of the 0.5 McFarland standard. The suspension density of the test organism was matched with that of the standard by adding more E. coli colonies or more sterile saline as needed. This was done to ensure that the bacterial suspensions had the appropriate density for the antibiotic susceptibility test, allowing for accurate and reliable results (Cheesbrough, 2010).
The antibiotic susceptibility test of the beta-lactamase-producing Escherichia coli strains was performed using the agar-disc diffusion method, per the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI, 2016). A panel of ten antibiotics was tested on these strains, which included ofloxacin (10 µg), pefloxacin (10 µg), ciprofloxacin (10 µg), augmentin (30 µg), gentamicin (10 µg), streptomycin (30 µg), ceporex (10 µg), nalidixic acid (30 µg), cotrimoxazole (30 µg), and ampicillin (10 µg). To conduct the test, 0.5 ml of the suspension of the test organism was placed in the middle of prepared Mueller-Hinton agar plates and spread evenly using a sterile cotton swab stick. Subsequently, a ring of each disc containing single concentrations of each antibiotic was placed on the inoculated surface using sterile forceps. The plates were then incubated at 37°C for 24 hours. After incubation, the zones of growth inhibition created by each antibiotic against the test organism were measured using a transparent metre rule, and the results were compared to the CLSI breakpoints (CLSI, 2016) to determine susceptibility, intermediate, or resistance levels.
A statistically significant relationship (P = 0.000) between the presence of beta-lactamase-producing E. coli strains in Kindirmo, Nasarawa town, and the different sampled areas using chi-square. The findings are detailed in Table 1.
The antibiotic susceptibility test results for the ten (10) beta-lactamase-producing E. coli strains are presented in Table 2. Among these strains, 70% were susceptible to ofloxacin, while 30% were susceptible to pefloxacin. However, all the beta-lactamase-producing E. coli strains were completely resistant to streptomycin, ceporex, nalidixic acid, cotrimoxazole, augmentin, and ampicillin.
The antibiotic resistance patterns of these ten beta-lactamase-producing E. coli strains are displayed in Table 3, with six different patterns observed, including combinations of 6, 7, and 8 antibiotics. No resistance pattern was found with a single, two, three, four, or five antibiotics.
Table 1: Occurrence of Beta-Lactamase-Producing Escherichia coli Strains in Kindirmo in Nasarawa Town, Nasarawa State, Nigeria
| Location | No. Collected | No. Positive | (%) Occurrence | P-value |
|---|---|---|---|---|
| TMH | 60 | 5 | 8.3 | |
| GNK | 50 | 3 | 6.0 | |
| FPC | 30 | 1 | 3.3 | 0.000 |
| NSM | 29 | 1 | 3.4 | |
| Total | 169 | 10 | 5.92 |
No. = Number, TMH= Tammah, GNK= Gunki, NSM= Nasarawa Market, FPC= Federal Polytechnic, Nasarawa Campus
Table 2: Antibiogram Susceptibility Profile of Beta-Lactamase-Producing Escherichia coli isolated from Kindirmo in Nasarawa Town, Nasarawa State
| N = 10 |
|---|
| Antibiotic Conc. (µg) R (%) I (%) S (%) |
| Ofloxacin 10 0(0) 3(30) 7(70) |
| Pefloxacin 10 5(50) 2(20) 3(30) |
| Ciprofloxacin 10 0(0) 10(100) 0(0) |
| Augmentin 30 10(100) 0(0) 0(0) |
| Gentamicin 10 5(50) 4(40) 1(10) |
| Streptomycin 30 10(100) 0(0) 0(0) |
| Ceporex 10 10(100) 0(0) 0(0) |
| Nalidixic acid 30 10(100) 0(0) 0(0) |
| Cotrimoxazole 30 10(100) 0(0) 0(0) |
| Ampicillin 30 10(100) 0(0) 0(0) |
N = number, Conc. = Concentration, R= Resistance, I= Intermediate, S= Susceptible
Table 3: The antibiotic Resistance Patterns of beta-lactamase-producing Escherichia coli from Kindirmo in Nasarawa Town, Nasarawa State
| No. of Antibiotics Resistance Pattern No. (%) of Strain |
|---|
| 6 AUG, STR, COT, NAX. CEP 1(10) |
| 6 GEN, STR, CEP, NAX, COT, AMP 1(10) |
| 7 PEF, GEN, STR, CEP, NAX, COT, AMP 1(10) |
| 7 PEF, AUG, STR, CEP, NAX, COT, AMP 3(30) |
| 7 AUG, GEN, STR, CEP, NAX, AMP 2(20) |
| 8 PEF, AUG, GEN, STR, CEP, NAX, COT, AMP 2(20) |
PEF= Pefloxacin, AUG= Augmentin, GEN= Gentamicin, STR= Streptomycin, CEP= Ceprorex, NAX= Nalidixic acid, COT= Cotrimoxazole, and AMP= Ampicillin
The study found that 10 (5.92%) of the 169 Kindirmo samples examined were contaminated with beta-lactamase-producing Escherichia coli strains. This could reflect poor sanitary practices employed during milking, milk handling, processing, and distribution. This percentage occurrence is lower than that recorded in some studies in India and Osun State, Nigeria (Khan et al., 2014; Abike et al., 2015). They recorded 20 and 9.2% occurrence of beta-lactamase-producing Escherichia coli strains in milk, respectively. The lower occurrence of beta-lactamase-producing E. coli strains in Kindirmo in this study compared to other studies could be attributed to the traditional methods of producing the product, which might have exposed it to fewer contaminants. The occurrence of beta-lactamase-producing E. coli in Kindirmo in this study reflects poor sanitary practices during milking, milk handling, and processing. However, this study emphasises the need for improved sanitary practices to further reduce bacterial contamination in Kindirmo.
As observed in this study, the complete resistance of the beta-lactamase-producing E. coli strains to several antibiotics is a significant concern. This resistance could be due to contamination from human sources or the environment, as these antibiotics are not commonly used in livestock management in the study area. The multidrug-resistant nature of the BLPEC strains is worrisome, as it can lead to the transmission of antibiotic-resistant bacterial pathogens to humans via the food chain (Badri et al., 2017; Akarsh et al., 2019; Kamaruzzaman et al., 2020; Nahar et al., 2023).
The six (6) antibiotic resistance patterns recorded in this study disagree with the three (17) and thirteen (13) patterns recorded among E. coli isolated from milk in Osun state, Nigeria (Abike et al., 2015). Differences in resistance patterns between this study and the others could be attributed to varying antibiotic use and misuse practices in different areas.
A 5.92% occurrence of beta-lactamase-producing E. coli strains was recorded in this study. This highlights the potential public health risk associated with the consumption of Kindirmo in the study areas. The beta-lactamase-producing E. coli strains isolated in this study were completely resistant (100%) to ampicillin, augmentin, cotrimoxazole, nalidixic acid, streptomycin, and ceporex. This is a cause for serious public health concern as these multidrug-resistant E. coli strains can be transmitted to humans through the food chain and cause infections that would not respond to chemotherapy, leading to an increased risk of death among affected individuals. It is recommended that basic hygiene requirements be met in preparing and selling dairy products. This will greatly enhance the overall quality standards and safety of the product. Also, relevant authorities should raise awareness among the general public about the dangers of indiscriminate antibiotic use. This will help reduce the emergence of antibiotic-resistant bacteria and promote responsible antibiotic consumption practices.
The authors have not declared any conflict of interest.
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