Identification of antibiotic resistance genes in Escherichia coli from subclinical mastitis milk in dairy cows and goats, East Java Province

Antibiotics are still used to treat mastitis in dairy cows in Indonesia. This study aimed to analyse antibiotic resistance genes in Escherichia coli (E. coli) from subclinical mastitis milk in East Java Province, Indonesia. The samples consisted of subclinical mastitis milk from cows and goats. A total of 592-quarter cow’s milk and 71 goat’s milk samples from both halves of the udder were collected from 67 farms in Lumajang, Banyuwangi, Malang, Sidoarjo, Jember, Pasuruan, Probolinggo, and Mojokerto. Subclinical mastitis samples were screened using the California mastitis test (CMT). E. coli was identified by phenotypic and genotypic methods. E. coli was confirmed with a primer specific to the polymerase chain reaction (PCR) technique. Gene resistance of E. coli was tested using the multiplex-PCR (mPCR) technique with primers encoding the genes temoneira enzyme (TEM), oxacillinase (OXA), sulfhydryl variable (SHV), and cefotaximase-munich IV (CTX-M IV). These genes were chosen because mastitis treatment generally uses oxacilline and β-lactam antibiotics. All data obtained were analysed descriptively. The results show that six isolates of E. coli (46.15%) carried a single resistance gene (TEM or SHV) and two isolates (33.33%) were confirmed as multiple drug-resistant organisms (MDROs) (TEM and SHV). The resistance genes were found in samples originating from Blitar, Banyuwangi, Lumajang, and Pasuruan Regencies. This research implies that antibiotic-resistance genes found in E. coli on certain farms are dangerous and may allow gene transmission to other bacteria that make treatment for mastitis or other bacterial infections ineffective.

East Java is a province with the highest dairy cow population in Indonesia.Based on data from Statistics Indonesia (2022), it is known that in East Java, the population of dairy cows is 314 385 from a total national 592 897 heads, and the population of goats is 3 897 185 from a total national 19 397 960 heads.This number increased by approximately 2% from the previ-ous year.Regrettably, the total milk production from dairy livestock in Indonesia was insufficient for national needs.Milk production in East Java was 543 687.16 tons and national production was 968 980.14 tons (Statistics Indonesia 2022).Based on the needs of the human population in Indonesia, this number is still very low.The average per capita milk consumption per year is 16.9 g. https://doi.org/10.17221/80/2023-VETMEDOne of the factors causing low milk production is the presence of disease (Biobaku and Amid 2018), especially mastitis.There are two forms of mastitis based on the physical symptoms.Clinical mastitis is udder inflammation accompanied by obvious physical symptoms, whereas subclinical mastitis is not (Kalinska et al. 2017).The prevalence of subclinical mastitis in East Java Province is very high, reaching 67% (Widianingrum et al. 2022).
Treating mastitis caused by E. coli is often difficult because it can produce extended-spectrum betalactamases (ESBL) enzymes (Prasetya et al. 2019).ESBL is a class of beta-lactamase enzymes located in plasmids or chromosomes that control genes producing lactamase (Masruroh and Sudarwanto 2016).These enzymes are the Temoneira enzyme (TEM), oxacillinase (OXA), sulfhydryl variable (SHV), and cefotaximase-munich (CTX-M) (Lai et al. 2022).Sierra et al. (2023) reported that due to these enzymes, E. coli is resistant to antibiotics.In Indonesia, dairy farmers still use antibiotics, so we hypothesised that resistant bacteria spread in various regions.In this study, we performed the resistance gene mapping in cases of subclinical mastitis caused by E. coli infection in East Java Province.

MATERIAL AND METHODS
The milk samples were obtained from lactating dairy cows, and each farmer carried out the milking according to their operational procedures.

Sampling of subclinical mastitis milk
The sample collection consisted of subclinical mastitis milk from cows and goats in East Java Province.
A total of 592 quarter cow's milk samples and 71 goat's milk samples from both halves of the udder were collected from 67 farms in Lumajang, Banyuwangi, Malang, Sidoarjo, Jember, Pasuruan, Probolinggo, and Mojokerto.Subclinical mastitis samples were screened using the California mastitis test (CMT).

Isolation and identification of Escherichia coli
Positive CMT samples were grown on selective media Eosin Methylene Blue Agar (EMBA) (Oxoid, Hampshire, United Kingdom) and tested for their biochemical properties.The non-resistance bacteria as a negative control in this study was E. coli BL21 from the Agrotechnology Laboratory, Faculty of Agriculture, University of Jember, Indonesia.Colony characters referring to E. coli were isolated from DNA using the DNA Extraction Kit (Geneaid Biotech, New Taipei City, Taiwan).A molecularbased determination for particular E. coli isolates was done using polymerase chain reaction (PCR) (Bio-Rad Laboratories, Hercules, CA, USA).Briefly, the DNA of E. coli was isolated following Windria et al. (2016) and subjected to a standard cycle PCR condition using E. coli species-specific primers with 16S rRNA primers (Table 1).
PCR mixture consisting of 10 μl PCR Master Mix 2X My Taq HS Red Mix (Bioline, London, UK), two μl primer reverse, two μl primer forward, four μl nuclease-free water, and two μl genomic DNA templates.PCR results were analysed on 2% agarose (Promega, Madison, USA) with FloroSafe DNA Stain two μl (Axil Scientific Pte Ltd, SCoence Park Road, Singapore).Four μl of PCR results and three μl of 100 bp ladder (Thermo Scientific, Lietuva, Lithuania) were placed into the gel wells and then electrophoresed with an electric current of 100 volts for 30 minutes.Electrophoretic DNA bands were observed using a UV transilluminator (Major Science Co., Ltd., Taoyuan City, Taiwan).

Antibiotic resistance gene mapping
For antibiotic resistance gene mapping, ArcGIS v10.8 (Environment Science & Research Institue, India) was used.

Data analysis
The data obtained were analysed and presented qualitatively using percentages, tables, and figures.

RESULTS AND DISCUSSION
Subclinical mastitis is diagnosed by the California mastitis test (CMT).Alkyl aryl sulfonate (a component of CMT) is highly sensitive to the number of nuclei of somatic cells or leukocytes.Somatic cells will react with CMT reagents, marked by the appearance of a gel (Sevitasari et al. 2019).The present study shows that subclinical mastitis cases in East Java are relatively high, reaching more than 60% in both cattle and goat milk samples.There were 397 from 592 (67.06%) cow's milk samples and 47 from 71 (66.19%) goat's milk positive for CMT.
Based on the microbiological a total of 13 (2.9%)(suspected) E. coli bacteria were found in cow and goat milk samples.To validate the bacteria, we confirmed it with the genotypic methods and we identified six E. coli strains in this study (Table 3, Figure 1).The E. coli was distributed in four areas of East Java province.Determination of the bacteria that cause mastitis is done by microbiological test, and confirmation can be done using PCR assay (Widianingrum et al. 2022).
There was no interaction between the CMT score and the presence of E. coli bacteria in subclinical mastitis cases in this study (Table 4).This is an important finding in our research because, at a low score (CMT + 1), E. coli bacteria were found which can harm livestock and milk consumers.E. coli bac- teria in subclinical mastitis milk in this study were found in Lumajang, Banyuwangi, Sidoarjo, and Blitar samples.This fact can be useful for farmers to treat their livestock diagnosed with mastitis immediately even though the CMT score is low (CMT + 1).We used 16S rRNA in this study because this gene is most frequently applied as a molecular marker compared to the other two types of ribosomal RNA, namely 5S rRNA and 23S rRNA.Generally, 16S rRNA is more stable and has a hypervariable region which is a particular area for identifying genus and species (Noer 2021).
Based on the distribution of antibiotic resistance gene data (Table 5, Figure 2), we found four (66.67%) isolates of bovine E. coli expressing TEM genes, and there were no other genes.In goat's milk, there were two isolates (33.33%) of E. coli that had the TEM gene, two isolates (33.33%) having the SHV gene, and two isolates (33.33%) having the TEM + SHV genes.These findings indicate that the E. coli bacteria that cause mastitis in our studied area have resistance traits that encode the TEM or SHV gene or multiple genes (TEM and SHV).Based on the international expert proposal for Interim Standard Definitions for Acquired Resistance, if there is an incidence of resistant isolates to at least one antibiotic from more than three groups of antibiotics, then multiple drug resistance organisms (MDRO) have occurred (Basak et al. 2016).Several pathogens, including MDROs, include methicillin-resistant S. aureus (MRSA), vancomycin resistance enterococcus (VRE), certain Gram-negative bacilli (GNB) including Enterobacteriaceae with plasmid encoded extended-spectrum beta-lactamases (ESBL).In the last decade, MDROs have shifted from Grampositive to Gram-negative bacteria.This is due to the emergence of Gram-negative bacteria resistant to various antibiotics (Bhattacharya 2013).    2 The distribution of resistance genes possessed by E. coli in this study is depicted in Figure 3.
Four samples carrying the TEM gene were found in the specimens isolated from Banyuwangi, Lumajang, and two others from Pasuruan.The inappropriate use of antibiotics in treating infected animals can increase the case numbers of antibiotic resistance (Kraemer et al. 2019).Low concentrations of antibiotics (subtherapeutic) can encourage genetic modifications that develop resistance (Ventola 2015).
The prevention step is to start mitigating MDRO infections.Farmers should be more careful in using antibiotics (e.g.efficiency of indication, dosage, duration, and use of antibiotics as medicine) (Ministry of Health of the Republic of Indonesia 2015).
However, using antibiotics for the treatment of mastitis also requires attention because of their residues in milk and meat.Usually, the minimum withdrawal time for antibiotics is five days after therapy, or 13 days for broad-spectrum antibiotics (Meutia et al. 2016).
In terms of numbers, our findings are in the minority.But, resistance genes can be transmitted, causing the spread of resistance traits (Batabyal et al. 2018).The environment acts as the origin of resistance.This is because only 10-80% of antibiotics are metabolised, and the rest is excreted as active compounds through urine and faeces into the environment, which can make environmental microbes resistant (Food and Agriculture Organization 2018).Humans, animals, and the environment are interconnected, and bacterial transfer, including mobile genetic elements (MGEs) (plasmids and transposons) between species is easy (Woolhouse et al. 2015).This study implies that dairy products from several farms can spread antibiotic-resistant pathogens so that they have the potential to transmit zoonoses (Khusnan et al. 2016).Therefore, information on the prevalence of resistance gene distribution is precious in understanding the distribution mechanism in several regions and as an evaluation of the mastitis treatment process carried out by breeders (Han et al. 2022).Infectious diseases caused by resistant bacteria result in prolonged illness, increasing the risk of death, failed treatment or livestock becoming a carrier (Humaida 2014).
In conclusion, it can be stated that resistance genes have been found in E. coli from milk infected with subclinical mastitis in goats and cattle in Lumajang, Banyuwangi, Sidoarjo, and MDRO in Blitar, East Java.The information provided in this research is critical to identifying gaps in knowledge that will guide future studies to develop therapeutic interventions for mastitis.

Table 1 .
Oligonucleotide primers coding E. coli and antibiotic-resistance genes

Table 3 .
Distribution percentage of E. coli isolated from cow's and goat's milk in East Java

Table 4 .
CMT results, phenotypic, and genotypic identification of E. coli

Table 5 .
Distribution of antibiotic resistance genes in cow's and goat's milk samples