Molecular identification of blaTEM and blaCTX-M genes in multidrug-resistant Escherichia coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia

Escherichia coli is a bacterial species that typically lives in both human and animal digestive tracts. However, under some conditions, E. coli can spread outside of the digestive system (21). Numerous studies have shown that milk is an advantageous medium for the growth of E. coli and that an overabundance of these bacteria in this foodstuff can be harmful to the general public’s health (18, 28). Antibiotic resistance has become a major issue on a global scale as a result of their use to treat diseases in humans and animals. Long-term antibiotic use will have an effect on the normal bacterial ecology, where pathogens adapt and change to survive. Escherichia coli is a bacterium that can easily gain an antibiotic deactivation enzyme expressed by an antimicrobial resistance gene (24). Penicillin, third generation cephalosporins, and monobactams are known to be hydrolysed by enzymes termed extended-spectrum beta-lactamases (ESBLs) from E. coli bacteria, these enzymes being the subject of extensive research at the moment.

The spread and transfer of E. coli with ESBLs can take place through food supply chains, contaminated faeces, contaminated water, and hazardous waste. Clinical symptoms or disorders caused by E. coli can appear as urinary tract infections, septic shock, and diarrhoeal illnesses (9, 18). Mastitis is a condition that can affect lactating animals and has been linked to E. coli infections (3, 12). According to research from 2019, 5.21% of samples of E. coli in dairy cattle faeces in Indonesia were ESBL-producing strains (19). Data from dairy cattle milk samples showed an incidence of ESBL E. coli of up to 2.15% in 2021 (2). Additional research into the prevalence of ESBL E. coli in 2021 on dairy farm samples indicated the rate to be up to 54% (17). However, it was estimated that by 2022 up to 0.18% of milk samples and the area around dairy cattle farms would have ESBL E. coli (28). The surroundings of dairy cattle sheds may contain various bacterial elements of antimicrobial resistance (27, 30). Mobile genetic elements can move between bacterial species, and transmission of resistance elements to other bacteria through plasmids and transposons can be accelerated and increased by animal activities and agricultural as well as human waste that pollutes the environment (11). An environment which is optimal in the aspect of providing bacterial resistance elements can be the main source from which these elements transfer to bacteria which could potentially infect humans, animals, or other environments (20).

Escherichia coli requires careful attention because of its strong ability to transmit resistance genes both within and between species (8). When E. coli with the capacity to produce ESBL enzymes infects people and other animals, it poses a serious threat. The ESBL enzyme in E. coli is encoded by several different ESBL genes, including the blaTEM, blaSHV, and blaCTX-M genes found in bacterial plasmids (29). Of these three, the blaCTX-M gene predominates in E. coli bacteria, and it can be co-expressed with the blaTEM gene to create the ESBL enzyme (15). A previous study discovered the phenomenon of the majority of the blaSHV and blaTEM genes becoming inactivated, making the blaCTX-M gene more widespread in E. coli bacteria (26). As noted above, the ESBL enzyme hydrolyses penicillin, third generation cephalosporins, and monobactams, meaning that three classes of antibiotic are resisted by bacteria producing the enzyme. Three antibiotic classes is the threshold for classification of a bacterial strain as multidrug resistant (MDR). Therefore, the objective of this study was to molecularly identify blaTEM and blaCTX-M genes among MDR Escherichia coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia.

In total, 110 milk samples were taken from 45 dairy farms in the Indonesian region of Tulungagung. The research was carried out from August to October of 2021. Samples were acquired from all four quarters and then placed into sterile sample vials, carefully capped, and chilled in the refrigerator for approximately 2 h before being taken to the laboratory for research. Samples were treated in the Veterinary Public Health Laboratory at the Faculty of Veterinary Medicine, Airlangga University, Indonesia. The isolated E. coli was incubated in GranuCult BRILA brilliant green lactose broth medium (cat. No. 105454; Merck, Darmstadt, Germany) medium at 37°C for 18 to 24 h. Eosin methylene blue agar (EMBA) selective was used to cultivate E. coli bacteria, which were then allowed to stand at warm temperatures (35–37°C) for 20–24 h. Colonies were verified using a Gram stain kit (cat. No. K001-1KT; HiMedia, Maharashtra, India) (9, 27). Biochemical analysis confirmed the presence of pure E. coli colonies by means of triple sugar iron agar (TSIA) (cat. No. 103915; Merck) and indole, methyl red, Voges–Proskauer and in citrate (IMViC) tests, the latter using sulphide indole motility (cat. No. 105470; Merck) and methyl red and Voges–Proskauer media (cat. No. 105712; Merck) and Simmons citrate agar (cat. No. CM155; Oxoid, Basingstoke, UK) (23, 28).

Antibiotic sensitivity testing on E. coli isolates was carried out using the disc diffusion method, as advised by the Clinical and Laboratory Standards Institute (5). After being prepared as instructed by the manufacturer, Mueller–Hinton agar (MHA) (cat. No. 105437; Merck) was cooled to 45–50°C and poured into plates. The medium was then allowed to solidify. The EMBA medium culture of E. coli isolates was grown for 18–24 h and standardised by dilution to 0.5 McFarland turbidity equivalence. A sterile swab stick was immersed in the standard E. coli dilution, dried to remove the excess load of inoculum, and smeared across the surface of the ready MHA plate. The MHA plates were given some time to dry with the lid closed at ambient temperature (29°C). The antibiotic discs for E. coli susceptibility testing (Oxoid, Basingstoke, UK) with a panel of tetracycline (30 μg), streptomycin (10 μg), chloramphenicol (30 μg), trimethoprim (5 μg) and aztreonam (30 μg) were carefully placed on MHA plates using sterile forceps.

To investigate ESBL production by E. coli isolates, the double-disc synergy test (DDST) was employed. Cefotaxime (30 μg), ceftazidime (30 μg) and amoxicillin-clavulanate (30 μg) were the antibiotic discs used in the DDST (Oxoid). After phenotypic confirmation with the DDST, MDR and ESBL E. coli were genotypically validated by further examining the presence of the ESBL enzyme–coding blaTEM and blaCTX-M genes using multiplex PCR as detailed in Table 1 (2, 19). The QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to extract bacterial DNA (23). As positive controls, Escherichia coli ATCC 35218 was utilised for the blaTEM gene and ESBL E. coli EQASIA 2021/E 21 for the blaCTX-M gene. Escherichia coli ATCC 25922 was the negative control. After amplification, the amplicons were subjected to UV light for the PCR product to be read on gel electrophoresis using a documentation system (Promega, Madison, WI, USA).

The study examined 110 milk samples total from 45 dairy cattle farms, of which 101 (91.82%) tested positive for E. coli based on the characteristics of the EMBA culture (Fig. 1), on Gram staining and on the TSIA and IMViC tests. The antibiotic susceptibility test conducted on the 101 isolates positive for E. coli showed that four (3.96%) were resistant to three or more antibiotics, as shown in Table 2. The E. coli isolates shown in Fig. 2 to have had resistance to three or more antibiotics were MDR. In this study, aztreonam resistance was discovered in two (1.98%) of the isolates. The evaluation of the DDST following incubation revealed synergy between cefotaxime/ceftazidime and the amoxicillin-clavulanate combination, as evidenced by an expansion of the inhibition zone by 5 mm between the disc diameters of the two antibiotics, which was indicative of ESBL-positive E. coli bacteria (23, 28). One (0.99%) ESBL-producing isolate was obtained through being confirmed positive on the DDST, which is presented in Fig. 3.

Fig. 1.

Fig. 2.

Fig. 3.

The four MDR isolates showed three patterns of antibiotic resistance, which are described in more detail in Table 3. The patterns were tetracycline (TE)-streptomycin (ST)-chloramphenicol (C) antibiotic resistance proven for two E. coli isolates, TE-ST-trimethoprim (W) antibiotic resistance observed for one E. coli isolate and TE-ST-W-aztreonam (ATM) resistance displayed by the last MDR isolate. The single ESBL-positive isolate of E. coli had resistance as TE-ST-W-ATM.

Molecular identification results showed that three (2.97%) MDR isolates confirmed DDST negative were detected to have the blaTEM gene and one (0.99%) isolate confirmed positive DDST was detected to have the blaCTX-M gene. Visualisation of the bands of the blaTEM and blaCTX-M gene fragments in this study is presented in Fig. 4. The electrophoresis results of positive isolates showed the same fragments as the positive controls, with gene lengths of 1,086 bp for the blaTEM gene and 550 bp for the blaCTX-M gene, while the results of isolates negative for the blaTEM and blaCTX-M genes did not represent the same fragments as the positive controls.

Fig. 4.

In many nations, MDR E. coli is highly prevalent and is the cause of serious and hard-to-treat infections (28). The risk of MDR E. coli infection is increased by milk contamination, which can be caused by improper sanitation practices during milking and milk processing, as well as by dairy cow contact with reservoir animals. Four (3.96%) of the 101 E. coli isolates in this study were confirmed to be MDR E. coli, which means they were resistant to three or more antibiotics. This percentage is lower than that noted in a previous study which found 9 (7.26%) MDR E. coli isolates out of 150 isolates (28). Another study of faecal samples from cattle suffering diarrhoea found 21 (77.8%) and 28 (63.6%) MDR isolates of E. coli from samples of dairy cows and beef cattle, respectively (31). Escherichia coli isolates were tested in Ethiopia and 27 (100%) were MDR, 188 (100%) E. coli isolates in Nigeria were reported to be resistant to three to seven different classes of antibiotics, and 76 (53%) E. coli isolates demonstrated multidrug resistance in Vietnam (4, 7, 14). A discovery of MDR E. coli was made in milk, perhaps because of improper or excessive antibiotic use in treating infectious diseases in dairy cattle, environmental and farm personnel contamination transferred during the milking process, or the free movement of the animal (25). In this study, only one (0.99%) MDR isolate of E. coli was found to be positive in a DDST.

Escherichia coli with ESBLs being present in milk is dangerous enough to warrant special attention, as this bacterium can harm human consumers and calves. When dairy calves are lactating, ESBL E. coli can sometimes be found in their milk, whether or not they are exhibiting mastitis symptoms. This suggests that inadequate cleanliness of the milking pens also poses a risk of ESBL E. coli contamination of cow’s milk products (25). It can be difficult to find alternative medicines to treat mastitis brought on by infection with ESBL E. coli, because many antibiotics (third-generation cephalosporins and aztreonam) are ineffective against such E. coli after the ESBL enzyme hydrolyses them (26). Animals can be responsible for human intestinal illnesses, and humans can contract them either directly from the animals (by eating food of animal origin, for example), or indirectly (via drinking water tainted with animal waste) (29). The animal-hosted pathogens causing these diseases in humans may transfer to people along similar exposure routes to those which have been described for various samples regarded as potential carriers of ESBL E. coli (17). The many potential pathways of E. coli ESBL transmission make epidemiological investigation very difficult (8). The expansion of ESBL-coding genes among different bacterial species will be facilitated through interactions at the microbial level in humans, animals, and the environment through horizontal gene transfer (2).

Molecular identification results showed that three (2.97%) MDR E. coli isolates confirmed ESBL-negative by DDST were detected to have the blaTEM gene and one (0.99%) MDR E. coli isolate confirmed ESBL-positive by the same test was detected to have the blaCTX-M gene. Bacteria that are positive in the DDST are ascertained to be ESBL enzyme–producing bacteria. The ESBL bacteria were all cephalosporin-resistant, and inhibitory zone interactions with beta-lactamase inhibitor medicines such as clavulanate were discovered (Fig. 3). Moreover, the carriage of ESBL genes by the isolates implied that they could act as reservoirs of antibiotic resistance. Foods of animal origin have regularly been reported to contain E. coli with the blaTEM and blaCTX-M genes (22). In this study, the findings of E. coli isolates containing the blaTEM and blaCTX-M genes are in accord with those of other reports, which showed the same prevalence of ESBL coding genes detected in milk samples (10) and environmental samples (6). It was found that E. coli with ESBL encoded by the blaCTX-M gene and also exposed to antibiotics may under certain circumstances be able to spread the gene to other pathogenic bacteria (9).

A study has shown that CTX-M gene–bearing E. coli is the dominant genotype, the gene often being seen singly or in combination in strains of this genotype (15). Other research has investigated K. pneumoniae which produces ESBL encoded by the blaCTX-M gene (20). According to a study, the blaCTX-M-15 gene was discovered in ten clinical isolates, the blaCTX-M-1 gene in two clinical isolates, the blaCTX-M-14 gene in two clinical isolates, and the blaCTX-M-9 gene in two food isolates (1). The blaCTX-M gene–bearing E. coli genotype is one of the most common ESBL genotypes that cause human infections in various countries (29). Since there is a strong correlation between the presence of ESBL E. coli in food and the development of infections in humans, it can be inferred that food of animal origin may contain resistant bacteria, aiding in their spread among humans.

The presence of ESBL E. coli in milk can be associated with the milking process and inadequate environmental sanitation (16). A major portion of milk contamination is caused by improper and unclean handling of milk, particularly during the milking process. Understanding and identifying the potential for limiting the spread of ESBL-coding genes and infection in people requires an integrated approach. Global cooperation in suppressing the ecology and thus the development of ESBL E. coli for the protection of public health can be achieved through a multisectoral approach to healthcare in the fields of veterinary medicine and animal food production (13). The implementation of the One Health integration idea is anticipated to hasten disease prevention and prediction in the fight against ESBL E. coli.

The discovery of the blaTEM and blaCTX-M genes in milk samples from dairy cattle farms in Tulungagung, Indonesia is concerning and requires prompt action to prevent antibiotic resistance from developing. Furthermore, this is a new potential threat of multidrug resistance which can spread and endanger public health. Multidrug-resistant bacteria can be encouraged to colonise milk by proximate sources of pollutants from dairy cattle urine and faeces. It is possible to keep the environment clean to prevent contamination from spreading extensively, and it is particularly important to do so in areas close to dairy farms. Additional risk factors for MDR E. coli dissemination, such as the usage of antibiotics and general dairy farm management, should be investigated in future studies. To inhibit the spread and rise in prevalence of MDR E. coli, in particular impeccable hygiene in milking processes must be guaranteed and more thorough wastewater treatment methods must be devised urgently. In order to prevent a large increase in the incidence of ESBL E. coli, it is essential to raise public awareness of the importance of sanitation and hygiene, and suitable initiatives should be state-directed. The One Health integrative approach might alternatively be applied as a prevention strategy if its implementation is continuous.