Full Length Research Article
Identification of BlaTEM Gene Encode Extended Spectrum Beta Lactamase (ESBL) Producing Escherichia coli Isolated from Fresh Beef
Lailatul Maghfiroh1, Mustofa Helmi Effendi2*, Wiwiek Tyasningsih3, Adiana Mutamsari Witaningrum2, Agumah Nnabuife Bernard4
Adv. life sci., vol. 11, no. 4, pp. 741-747, November 2024
*- Corresponding Author: Mustofa Helmi Effendi (mheffendi@yahoo.com)
Authors' Affiliations
2. Department of Veterinary Public Health, Faculty of Veterinary Medicine, Airlangga University – Indonesia
3. Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Airlangga University – Indonesia
4. Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki – Nigeria
[Date Received: 11/03/2023; Date Revised:17/07/2024; Date Published: 15/10/2024]
Abstract
Introduction
Methods
Results
Discussion
References
Abstract
Background: The community needs fresh beef of good quality. Dirty sanitation causes Escherichia coli to easily contaminate fresh meat and E. coli is one of the bacteria that can produce ESBL enzymes that impact human health. This study aimed to isolate Escherichia coli from fresh meat at the Surabaya Traditional Markets to identify the presence of the ESBL-encoding blaTEM gene.
Methods: One hundred fifty fresh beef samples were collected from six traditional markets located in Surabaya city. Then, a polymerase chain reaction (PCR) test was performed to detect the blaTEM gene which codes for ESBL in isolates that unveiled positive results for MDR (resistant to three antibiotics) and suspected beta-lactamase enzyme (resistant to ampicillin).
Results: The research showed that from 150 samples of fresh beef at the Surabaya Traditional Market, 68 Escherichia coli isolates were found (45.3%). Escherichia coli isolates were shown to be susceptible to the antibiotic’s ampicillin (73.5%), gentamicin (95%), tetracycline (7.5%), aztreonam (100%), and ciprofloxacin (95%). The highest percentage of resistance of Escherichia coli isolates to the antibiotic ampicillin (19.1%). PCR analysis of ampicillin-resistant isolates showed that the blaTEM gene encoding ESBL was present in 7 (53.8%) of the 13 Escherichia coli isolates.
Conclusion: The research results show that there is a risk of ESBL transmission to the community through Escherichia coli which was identified in fresh beef at the Surabaya Traditional Market. Therefore, it is hoped that public awareness can increase regarding food safety issues which can have an impact on public health.
Keywords: Escherichia coli; blaTEM; ESBL; Fresh beef; Public health
In the twenty-first century, antimicrobial resistance (AMR) is one of the greatest public health problems due to its widespread impact on humans, animals, and the environment [1,2]. Antimicrobial resistance (AMR) is a worldwide issue that poses a threat to human health because of inadequate research, dedication, infection control, illogical use of antibiotics, low-quality antibiotics, and inadequate supervision [3,4]. The emergence of pathogenic bacteria resistant to antibiotics causes an increase in patients infected with more difficult bacteria, takes longer to heal, and requires new types of antibiotics [5,6].
Beef is one of the important sources of animal origin for the community, especially to meet the needs of animal protein. Meat that contains complete nutrients makes it a good medium for the growth of bacteria, one of which is Escherichia coli. The presence of Escherichia coli in meat exacerbates the incidence of infection in humans because it can transmit infection and disease during processing, preparation or when consumed by consumers [7]. Fresh meat sold in retail markets can transmit Extended Spectrum Beta-Lactamase (ESBL) from animals to humans [8]. Research by Ansharieta et al., [9] showed that the TEM gene encoding ESBL was found in foodstuffs of animal origin. This is also confirmed by Imasari [10] that the gene encoding ESBL has spread between humans and animals. Of the ESBL-positive isolates, 48.1% had the TEM gene identified [11]. blaTEM is one of the most frequently confirmed ESBL coding genes in a study [12]. The research of Naelasari et al., [13] also showed that the TEM gene was found in isolates of patients from Dr. Soetomo Hospital. As per the findings of Bajpal et al., [14], the TEM gene accounted for 48.7% of the ESBL coding genes detected in isolates obtained from individuals suffering from urinary tract infections.
The increasing incidence of ESBL has become a public health focus. However, data on the incidence of ESBL produced by Escherichia coli in food of animal origin, especially fresh meat in the city of Surabaya, is still limited. No molecular research has detected the spread of the gene encoding ESBL resistance (blaTEM) produced by Escherichia coli isolated from fresh beef sold in traditional markets in Surabaya. Given the foregoing context, it is critical to carry out investigations to ascertain whether Escherichia coli isolates from fresh beef offered in Surabaya's traditional markets carry the blaTEM gene, which codes for ESBL.
Ethical approval
Since this investigation used fresh beef, ethical approval was not required. In Surabaya, Indonesia, six traditional markets provided samples of fresh meat.
Isolation and Identification of Escherichia coli
This research was conducted from February to April 2022. As many as 150 fresh beef samples were obtained from six traditional markets in the city of Surabaya, namely Wonokromo Market, Keputran Market, PacarKeling Market, Pabean Market, Manukan Market, and Jagir Market. The samples were isolated through a swab using a sterile cotton bud and planted in Buffered Peptone Water (BPW) media which functions as a resuscitation medium. They were transported using a cool box to the Division of Bacteriology and Mycology Laboratory, Department of Microbiology, Faculty of Veterinary Medicine, Airlangga University. Furthermore, 1 ml of each of the samples was planted into 9 ml of selective media enrichment Brilliant Green-bile Lactose Broth (BGLB), and coliform positive results were grown onto selective media of Eosin Methylene Blue Agar (EMBA) (Figure 1). The presumptive results of Escherichia coli were indicated by the growth of metallic green colonies with black midpoints. Furthermore, identification was carried out using Gram stain and IMViC biochemical test. Samples that yielded Escherichia coli were subjected to antibiotic sensitivity tests to determine their antibiotic resistance profile.
Antibiotic Sensitivity Test
Antibiotic sensitivity tests were performed on Escherichia coli isolates following the National Committee for Clinical Laboratory Standards [15]. To find out which five classes of antibiotics could stop the isolated Escherichia coli isolates from growing, the isolates were grown on Muller Hinton Agar (OXOID CM0337) medium and were given 30 µg of Aztreonam, 10 µg of Ampicillin, 30 µg of Tetracycline, 10 µg of Gentamycin, and 5 µg of Ciprofloxacin. The suspension of Escherichia coli isolate adapted to Mac Farland 0.5 standard was spread evenly on the surface of MHA media using sterile cotton buds and 5 different antibiotic groups were planted with a distance of 30 mm. There are three criteria, namely Susceptible (S), Intermediate (I), and Resistant (R). Furthermore, the sample isolates that showed positive results of MDR (resistance to three antibiotics) and presumptive Beta Lactamase enzyme (resistant to Ampicillin) were analyzed for the presence of blaTEM gene encoding ESBL using Polymerase Chain Reaction (PCR).
Molecular detection
The objective of the DNA extraction procedure is to remove the DNA from the bacterial cell before it is amplified in the PCR reaction. The DNA extraction used in this study followed the procedure for Extracting Gram-negative DNA from QIAGEN KIT.
PCR amplification of the blaTEM gene with 21 L of PCR reagents contained 12.5 L of PCR Master Mix, 1 L of forward primer, 1 L of reverse primer, 1 L of DNA template, and 5.5 L of RNAse free water. The mixing of PCR reagents was carried out using a thermocycler. The primers used and the PCR conditions are shown in Table 1 below.
5 L of PCR product was put into a 2% agarose gel well and electrophoresed at 100 V for 40 minutes in tris-acetate buffer. Afterward, the image was produced using ethidium bromide staining, UV illumination, and gel documentation equipment. A 100 bp DNA ladder served as the PCR marker. The results obtained in the form of a DNA band pattern with a size of 1086 bp were declared positive for the ESBL-encoding blaTEM gene.
Statistical Analysis
Following qualitative processing, data from molecular analyses, bacterial sensitivity tests, and isolation results are presented descriptively.
The Presence of Escherichia coli in Fresh Beef
The results of this study indicate the presence of Escherichia coli bacteria in fresh beef at the Surabaya City Traditional Market by 45.3% (68/150) as shown in Table 2.
Antibiotic resistance pattern
Sixty-eight isolates of Escherichia coli tested with antibiotic sensitivity showed that all Escherichia coli isolates were still sensitive to the antibiotic Aztreonam, namely 68 isolates (100%). Then, there were 13 resistant isolates, 5 intermediate isolates, and 50 sensitive isolates on the antibiotic ampicillin. In tetracycline antibiotics, there were 51 sensitive isolates, five intermediate isolates, and 12 resistant isolates. Furthermore, on the antibiotic Gentamycin, there were 61 sensitive isolates, three intermediate isolates and four resistant isolates. Ciprofloxacin isolates contained 61 sensitive isolates, three intermediate isolates and four resistant isolates (Table 3).
Molecular detection
PCR testing was used to find the ESBL-encoding blaTEM gene in isolates of Escherichia coli bacteria that were resistant to the antibiotic ampicillin. The results showed that of the 13 isolates of Escherichia coli isolated from fresh beef at the Surabaya Traditional Market, seven isolates (53.8%) had the blaTEM gene encoding Extended Spectrum Beta Lactamase (ESBL). The results of electrophoresis of PCR products, the blaTEM gene will show a DNA band of 1086 base-pair (bp) as shown in Figure 2.
Figures & Tables
One of the bacteria that play a role in AMR cases is Escherichia coli. Escherichia coli plays an important role in ecology and is used as a bioindicator of antimicrobial resistance [22]. This study showed higher results than the research of Eyi [16] which showed the prevalence of Escherichia coli isolated from beef was 25% (14/56) and research by Langgar et al., [17] showed a positive result of 9.75% Escherichia coli (4/21).
The prevalence of ESBL-producing Escherichia coli in fresh beef in Singapore is 7.3% and in Australia 5.3% [18]. However, previous studies have also shown higher results, such as the Sulaxono study [19]; namely from 189 fresh meat samples in the Maros Veterinary Centre Work Area, it was found that 85.19% (161/189) were contaminated with Escherichia coli. Research by Indraswari et al., [20] found 84.69% (83/98) of Escherichia coli in beef in the Yogyakarta. In Adzitey's research [21] Escherichia coli was successfully isolated from beef in abattoirs by 88%.
In order to acquire resistance genes from other bacteria and transfer their resistance genes to other bacteria, Escherichia coli functions as both a donor and a recipient of resistance genes. Therefore, in both human and veterinary medicine, Escherichia coli is considered to be the primary reservoir of resistance genes that cause treatment failure [23]. Given that it generates the Enzyme Extended Spectrum Beta-Lactamase (ESBL), Escherichia coli has the potential to be the primary source of AMR and is crucial to the development and spread of AMR pathways [1]. of except for cephamycin and carbapenems, the enzyme Extended Spectrum Beta-Lactamase (ESBL) may hydrolyze penicillin antibiotics as well as third- and fourth-generation cephalosporins and monobactams [24]. The global spread of Escherichia coli producing ESBL enzymes poses a significant threat to the effectiveness of antimicrobials, especially third and fourth-generation cephalosporins [25].
Where the difference in the prevalence of Escherichia coli according to Abayneh et al., [26] can be caused by differences in sanitation standards from the kiosks of fresh meat traders, low sanitation standards, and poor hygiene practices when handling meat contribute to cross-contamination of Escherichia coli isolates in meat. According to Haile et al., [27] contamination of beef or beef carcasses can occur during the preparation process such as skinning, removal of fat, and distribution. Furthermore, the area in traditional markets where beef is sold is still mixed with vendors selling other necessities; the meat that is placed on tables is not sterile, is left exposed without a cover, and is left to hang at room temperature; additionally, the traditional market setting, with its environmental conditions and all buying and selling activities carrying some risk of contamination. Fecal infection is one possible cause of ESBL-producing Escherichia coli in fresh meat. This is evidenced by the research of Palmeira et al., [28] that found Escherichia coli producing ESBL in the feces of beef cattle in Brazil. In Germany, 11.2% of beef cattle also have ESBL-producing Escherichia coli [29]. In addition, research by Montso et al., [30] revealed that there was ESBL-producing Escherichia coli in isolates from fresh meat and beef cattle in South Africa.
Aztreonam is a monobactam antibiotic that is used as an index for suspected ESBL. The study results showed that all Escherichia coli isolates were still sensitive to the antibiotic Aztreonam and was in line with exploration by Nadira et al., [31] who set up that Escherichia coli isolates from cow feces samples were still 100% sensitive to Aztreonam. The rare use of the antibiotic Aztreonam in beast is the reason that aztreonam perceptivity is still high. Still, these results are different from the previous study by Ansharieta et al., [32] which showed that Escherichia coli isolates from fresh cow's milk in East Java endured resistance to Aztreonam of 2.16%.
Ampicillin is a penicillin-class antibiotic that's used as an indicator of the presence of beta-lactam enzymes. The results of the study showing that 19 Escherichia coli isolates were resistant to Ampicillin are substantiation of a fairly high position of resistance and are in line with former exploration that more than 50 of beta- lactam antibiotics are resistant to bacterial isolates forming from food of beast origin. Exploration by Dsani et al., [33] set up that 57 of Escherichia coli isolates from beef were resistant to Ampicillin.
Antibiotics made from Ampicillin and Penicillin are constantly used to treat bacterial infections in livestock raised for food, like cattle. It's known that the antibiotics Ampicillin and Penicillin are frequently used to treat bacterial infections in animals similar to cattle. In addition, penicillin antibiotics in humans are also frequently used in the treatment of infections similar to urinary tract infections. Thus, while choosing a course of remedy for ails brought on by Escherichia coli that has been linked to beef, this needs to be taken into consideration. Ampicillin is a beta-lactam drug that works by inhibiting bacterial wall synthesis. Beta-lactam antibiotics bind to Penicillin-Binding Proteins (PBPs) in bacteria, thereby inhibiting bacterial cell wall synthesis because the trans-peptidase process between peptidoglycan chains is disrupted, then proteolytic enzyme activation occurs in the cell wall. Ampicillin resistance can occur by several factors, one of which is the formation of the beta Lactamase enzyme which is excreted by Gram-negative bacteria into the periplasmic cavity between the cytoplasmic membrane and the bacterial cell wall so that it can reach the right antibiotic target to interfere with the way the antibiotic works [34].
Inappropriate and excessive use of antibiotics causes selective pressure that favours the growth of resistant bacteria. Through conjugative plasmid-mediated horizontal gene transfer, the colonization of resistant bacteria in the intestines of people and animals result in the transmission of bacterial resistance genes in the intestinal flora. The majority of plasmid genes encode ESBLs. It was discovered that Enterobacteriaceae possessed plasmids containing resistance genes. The blaTEM gene is located on a plasmid [35]. The IncFII plasmid group is known as the group encoding the ESBL gene and is widely distributed in Enterobacteriaceae and is called the epidemic-resistant plasmid group. blaTEM is one of the dominant ESBL genes in the world and was found in human, animal, and environmental isolates. The blaTEM gene was found mainly in isolates of Escherichia coli and Klebsiella pneumoniae bacteria [36]. This result is higher than research by Dsani et al., [33] which detected the blaTEM gene by 4% (4/98) in Escherichia coli isolates isolated from fresh beef in Ghana.
The most prevalent beta-lactamase seen in Gram-negative bacteria is the blaTEM gene. 90% of Escherichia coli's resistance to ampicillin is brought on by the blaTEM gene production [37]. The presence of resistant bacteria in food-producing animals and food products of animal origin is caused by the continuous use of antibiotics for therapeutic and non-therapeutic purposes in the production system [38–40]. In addition, fresh beef can be contaminated with antibiotic-resistant pathogens at the point of unhygienic slaughter and sale. Although selective pressure is the main cause while, sanitary conditions at slaughter, sale, and processing sites can influence the intensity pattern of distribution along the food chain [33].
Fresh beef containing ESBL-producing Escherichia coli poses a major risk to public health, and people need to be made aware of the risks these pathogenic bacteria represent. The evidence by molecular identification using PCR in this study showed the presence of the blaTEM gene in fresh beef sold at Traditional markets in Surabaya. This proves that there is a risk of transmission of AMR caused by Escherichia coli producing ESBL from food of animal origin to humans.
Acknowledgements
This study was supported in part by the Lembaga Pengelola Dana Pendidikan (LPDP) Kementerian Keuangan Republik Indonesia with grant number: KET-3889/LPDP.4/2020.
Author Contributions
Lailatul Maghfiroh and Mustofa Helmi Effendi: Conceived, designed, and coordinated the study.
Wiwiek Tyasningsih: Designed data collection tools, supervised the field sample and data collection, and laboratory work as well as data entry.
Adiana Mutamsari Witaningrum: Contributed reagents, materials, and analysis tools.
Agumah Nnabuife Bernard: Carried out the statistical analysis and interpretation and participated in the preparation of the manuscript.
All authors have read, reviewed, and approved the final manuscript.
The author declare that there is no conflict of interest regarding the publication of this paper.
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