Isolation and Identification of Infection-Causing Bacteria in Dairy Animals and Determination of Their Antibiogram

Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture, Lanzhou, China Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou, China Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou, China West China School of Nursing, Sichuan University, Chengdu, China Department of Microbiology, University of Balochistan, Quetta 87300, Balochistan, Pakistan


Introduction
Dairy animals have always been one of the most studied animal species in terms of animal welfare [1], Nonetheless, there are important welfare issues that have yet to be resolved. Early weaning, which is still a common practice in the dairy business, has been one of those difficulties for the previous few decades [2].
Toxic metabolites from several pathogenic organisms living in milk and milk products may be present. Consumers get food poisoning when they eat such goods that are contaminated with these metabolites. Foodborne infection, on the other hand, is caused by ingesting viable pathogenic bacteria together with the food product [3]. e most common disease-causing bacteria in the milk are Mycobacterium bovis, Salmonella spp., Corynebacterium spp., Coxiella burnetii, Clostridium perfringens, Brucella, Staphylococcus spp., Yersinia enterocolitica, Campylobacter jejuni, Mycobacterium avium, Escherichia coli, Listeria spp., and coliforms [4,5], as Gramnegative opportunistic environmental bacteria, have been classified. Mastitis caused by E. coli is typically sporadic, with symptoms ranging from mild to severe and even fatal [6].
Meal-borne infection, on the other hand, is caused by ingesting viable pathogenic bacteria along with the food [3]. Total coliforms, E. coli, and other enteric bacteria in food, water, milk, and other dairy products in typically unclean conditions are reliable indications of fecal contamination. e presence of enteropathogenic and/or toxigenic microorganisms, which could pose a public health risk, is indicated by the recovery of E. coli from food [7]. According to public health agencies, these microbes are frequently linked to foodborne disorders and outbreaks [8].
e presence of these pathogenic bacteria in milk has become a major public health problem, particularly among individuals who continue to drink unpasteurized raw milk [9].
Antimicrobial drugs are used medically in animals and humans to treat bacterial infections, and they can also be added to commercial livestock and poultry feed at subtherapeutic dosages to promote growth [10]. During the twentieth century, antibiotics significantly reduced mortality associated with infectious diseases; however, their widespread and repeated use in animal farming has resulted in the emergence of bacterial multidrug resistance (MDR); as a result, the presence of antibiotic-resistant populations transforms infections that were once treatable into potentially life-threatening events. Antibiotics slow the growth of organisms without resistance mechanisms, but they have no or little effect on resistant infections, allowing them to survive and flourish in the host [11]. e purpose of this research was to determine the existence of infecting microorganisms and their antibiotic resistance patterns in the dairy farm environment, which could pose veterinary and public health risks by infecting animals and contaminating milk, water, and feces with antimicrobial-resistant avian strains as a result of poor milking hygiene.

Sample Collection.
Samples were collected from different potential points considered to be associated with contamination (critical sampling points). e sampling points were the teat during milking, drinking water trough of lactating animals, and fecal samples. Overall, 150 samples were analyzed.

Milk Samples.
A total of 60 milk samples from teats were collected from 20 cows and 15 buffaloes (30 hind and 30 teat). Milk samples were collected into sterile vials after washing, drying, and swabbing of teat ends with 70% ethyl alcohol and discarding of the first 3-4 streams of milk.

Drinking
Water. 30 water samples (50 ml from each farm) were collected from the drinking troughs of the lactating animals into disposable sterilized test tubes. All samples were stored in an ice box until transported to the laboratory.

Fecal
Samples. 60 fecal samples were collected from apparently healthy animals which showed no symptom of illness. Samples were collected with the use of sterile swab stick. e swab was transfer to its care, labeled, and taken to the laboratory.

Bacterial Isolation and Identification
2.2.1. Primary Culture. Nutrient broth, nutrient agar, MacConkey agar, eosin methylene blue (EMB) agar, Salmonella/Shigella agar, mannitol salt agar (MSA), and blood agar media were prepared, and swab samples were cultured aerobically and anaerobically over these media and incubated at 37°C for 24 h. Following, the incubation colony characteristics were observed, and smears were prepared, for Gram's staining for cell morphology.

Subculturing and Identification.
Purification of preliminary identified colonies was done by subculturing the isolated colonies on respective differential and selective media and repeated it several times. e purity of the samples was checked by examining stained smear. e pure culture was grown on agar slants, incubated at 37°C for 24 h, and stored at 4°C. e bacterial isolates were finally identified based on colony characteristic, microscopic morphology, biochemical properties such as catalase, oxidase, indole, coagulase, triple sugar iron, and Simmons Citrate Agar, and analytical profile index.
Round raised circular colonies on nutrient agar with Gram-positive reaction and round shape with bunches appearance under the microscope were presumed as Staphylococcus aureus and subcultured over MSA. Yellow colonies with catalase-positive reactions confirmed the presence of S. aureus.
Circular raised colonies with pink appearance on McConkey agar and rod-shaped bacteria with Gram-negative rection were subcultured over EMB agar, and the lactose-fermented colonies with green metallic sheen were considered and as conformed E. coli and subjected to analytical profile index for compete conformation.
Salmonella and Shigella spp. were subcultured on Salmonella/Shigella agar (SS agar), a Salmonella and Shigella spp.-specific agar. Typical colonies, such as Shigella, were detected as transparent or translucent colorless colonies on growth plates. e organisms were further confirmed with the help of Gram reactions, biochemical tests, and analytical profile index.

Multiple Antibiotic Resistance Index (MARI)
Determinations. MARI was determined using the formula MARI � a/b, where "a" represents the number of antibiotics that were successfully resistant and "b" represents the total number of antibiotics employed in the study. An isolate with a MARI value of 0.2 > indicates the presence of numerous antibiotic-resistant bacteria, as well as highly resistant bacteria [14].

Statistical Analysis.
e graphical representation was performed using the program Microsoft Office Excel, 2007. Descriptive statistics of the data obtained from the study are given with mean, standard deviation, frequency, and percentage analysis.

Isolated Bacterial Species.
Dairy farms included in this study raised from 10 to 50 animals (cows and native buffaloes). Cow's age ranged from 10 years, while buffaloes were around. Overall, four bacterial targets were identified in different sampling collection points in this study. e bacteria identified and their prevalence rates were Escherichia coli 88 (58%), Salmonella spp. 32 (21%), Shigella spp. 44 (29%), and Staphylococcus aureus 81 (54%).
ese are indicative of significant contamination of different sampling points. e most prevalent organism overall was E. coli, followed by S. aureus, Shigella spp., and Salmonella spp. In this study, the contamination degree of milk by the pathogenic bacteria is utterly worsened at each critical sampling point. High contamination level was observed at milk and fecal samples of cattle and buffaloes, while the least contamination level was observed in trough of lactating animals. e difference in isolation rate across different farms (critical sampling points) is statistically significant in E. coli and S. aureus, as given in Table 1 and Figure 1.

Antimicrobial Susceptibility of the Bacterial Isolates.
Antibiotics were found to have the best efficiency against both Gram-negative and Gram-positive bacteria, according to the antibiogram. Resistance to antibiotics appeared from the environmental E. coli, Salmonella spp., Shigella spp., and S. aureus isolated from teat milk, drinking water, fecal swab, and trough lactating animal. All the 4 bacterial isolates displayed resistance against 15 antibiotics. ese are indicative of significant contamination of different resistance rates. e most prevalent organism overall was E. coli, and S. aureus was observed with high resistance rate against antibiotics (Tables 2 and 3). e findings of this investigation clearly reveal that E. coli and S. aureus were resistant to all antimicrobials tested. E. coli isolates had the highest resistance rate, and S. aureus isolates were shown to be multidrug resistant ( Figure 2). Salmonella spp. and Shigella spp. showed lower resistance rate to all antimicrobials tested (Figure 3).

Determination of Multiple Antibiotic Resistance Index (MARI) Percentage.
Multiple antibiotic resistance index phenotypes were created by dividing the number of antibiotics resistant by the total number of antibiotics tested for isolates that showed resistance to three or more antibiotics (Table 4).

Resistance Rate with Different Bacterial Isolates.
When the zone diameter resistance was observed with 15 different antibiotics against most Gram-positive and Gramnegative bacteria, 15 antibiotics were found resistant for E. coli and S. aureus, for Shigella, 12 antibiotics were found to be resistant, and for Salmonella, 11 antibiotics were found to be resistance, as given in Table 5. e mean ± SD zone areas for the greater resistance for E. coli and S. aureus is already known to be multiresistant, followed by Salmonella and Shigella when the zone areas are for the low resistance, and there was little difference between mean ± SD zone areas for S. aureus and E. coli as given in Table 6.

Discussion
e small-scale dairy farming is a significant economic sector for the enhancement of agriculture and livelihoods in developing countries [15]. Milk samples, fore and hind's teats, cattle and buffaloes, fecal matter of 60 of dairy resident animals, and 30 of trough lactating waterfowls contained E. coli, Salmonella spp., Shigella spp., and S. aureus. Other researcher found that the practice of dairy animals in open yards and accumulation of their manure lead to the attraction of wild birds into dairy farms [16]. Similar studies isolated identical E. coli strains from excreta of wild birds collected from two dairy farms with 32.5 km distance apart, on the same sampling time [17,18]. e outcome of our study revealed that 120 of the total samples were contaminated accounting for large proportion. Escherichia coli and Staphylococcus aureus were the most common species in this study. is was followed by E. coli, Salmonella spp., Shigella spp., and S. aureus, with isolation rates of 88 (58%), 32 (21%), 44 (29%), and 81 (54%), respectively. In recent time, it was demonstrated that Escherichia coli was the predominant species in healthy Journal of Food Quality 3 animals; this finding is also in tandem with the current study [19]. Four bacterial species were prevalent from skin wound samples: E. coli, S. aureus, Salmonella, and Shigella spp. is reported that E. coli, Shigella spp., S. aureus, and Salmonella spp. and their occurrence percentages tandem with the current study [20]. In a similar manner, high rate of   Escherichia coli was reported in cows from Jordan [21]. ese findings are also consistent with other reports, where Escherichia coli was observed as the predominant species in healthy animals [19]. Various workers throughout world did high rate to isolate the bacterial organisms from wound samples of animals. However, the results of the present figures can be compared with the results of [20,22,23].
Handling of the animals, misuse of antibiotics, and other factors might be responsible for the differences certainly and not only geographical location. Overall, the high prevalence of E. coli and S. aureus occurrence over other isolates can be explained by the members of normal flora in animals; however, occurrence of E. coli, Salmonella spp., Shigella spp., and S. aureus is a pointer to high burden that have potential risk to animals and human health.
In this study, the disc diffusion method of detection was employed, previously reported as the method of detection. e major finding in the present study is the presence of multiple drug-resistant E. coli in dairy animals. In present study, ampicillin had the highest resistance rates in E. coli (56.8%), followed by ciprofloxacin (26.1%) and augmentin (25%). Antibiotic resistance among bacteria, particularly Escherichia coli isolated from cattle and other animals, is developing at an alarming rate, according to findings in other studies [24]. Resistance to doxycycline was the strongest in E. coli. In comparison, ampicillin (100%) and  Journal of Food Quality amoxicillin (100%) had a far greater resistance rate (42.11%) [25]. e major finding is the presence of multiple drugresistant E. coli in animals to commonly used antibiotics such as ampicillin (95%), augmentin (91%), cefotaxime (85%), tetracycline (72%), gentamicin (66%), and amoxicillin + clavulanic acid (58%) [26]. In this report, however, the highest rate of resistance to amoxicillin was found in Salmonella spp. (50%). In previous studies from Ethiopia, several researchers discovered antibiotic-resistant Salmonella isolates from milk [27,28] and also from other countries [29]. Tajbakhsh et al. reported that Salmonella spp. isolates were resistance to ampicillin (42.58%), tetracycline (42.58%), and nalidixic acid (78.57%) [30]. Addis et al. reported a high resistance rate of Salmonella isolates to ampicillin (100%) [31].
In this study, ampicillin had the highest resistance rate for Shigella spp. (36.3%), followed by cefpodoxime and ceftazidime (22.7%). Sanaa et al. found that Shigella isolates from raw milk were responsive to gentamycin (64.3%) and     [32]. e findings of this investigation clearly demonstrated that S. aureus was resistant to all antimicrobials tested, with the exception of ampicillin and ciprofloxacin. ese findings suggest that the problem is widely spread and distributed. Furthermore, overall resistance of S. aureus isolates to ampicillin, ciprofloxacin, augmentin, amoxicillin, and erythromycin was less than 25%, which is consistent with Ma et al.' study from a Taiwanese dairy farm. It is probable that these antimicrobials were less resistant since they are not often used in veterinary and possibly human medicine in the study area. Calderón-Jaimes et al. reported that the development of antimicrobial resistance is nearly always as a result of repeated therapeutic and/or indiscriminate use of them that gave similar suggestion [33]. However, the current investigation has revealed that S. aureus' resistance to routinely used antimicrobials (penicillin-G and tetracycline) is alarmingly high in the studied area. is is because tetracycline and penicillin are commonly and ineffectively used antimicrobials in animal and human medicine. e findings matched those of previous research conducted in other nations [34,35], suggesting that extended and indiscriminate use of some antimicrobials could lead to the development of resistance. is is in contrast to the findings of [36]. e multiple antibiotic resistance index (MARI) of the isolates recovered in the present study indicates multidrug resistance in nature. e MARI value > 0.2 is suggesting multidrug resistance due to high-risk application and contamination of antibiotics [37]. An average of 0.8 MARI in this study is higher than the report of [38]. High reported pattern MARI of 0.11-0.78 is reported from Ghana. ese findings demonstrate that the cattle were exposed to multiple classes of antibiotics. is can be explained by the sample size and antibiotic regulation in the study area, among other factors. Many other factors are involved.
When the zone diameters were multidrug resistance (mean ± SD) against bacterial organisms used in the study, it was determined that there was little statistically significant difference. It was determined that (±9.76 SD and ±9.75 SD) were resistance to S. aureus and E. coli, followed by Salmonella and Shigella, when the zone areas for the low resistance are (±9.38 SD and ±4.86 SD). ese findings determined that there was a little difference between S. aureus and E. coli. Various workers throughout world did high-rate antibacterial activity against S. aureus (zone of inhibition 8.0 mm), but less active against E. coli [39]. In the study results, S. aureus showed 10 mm zone inhibition, while there was no indication of inhibition against E. coli. e results of the study are consistent with the results of other studies [40]. In addition, on the 5th day of the application, somatic cell increase was observed due to the inflammatory reaction [41].
As a result, we recommend using molecular approaches to characterize these isolates for the presence of antibiotic resistance determinants, which could give data to back up our findings. Because E. coli and S. aureus are commonly found in animals, S. aureus found in milk, feces, and water of dairy cows and buffaloes could have come via human transmission, raising doubts about the hygiene standards used.

Conclusion
e study showed that the different sampling point matter of dairy animals could be considered as a potential risk factor for disseminating pathogenic in dairy animals, leading to contaminating the teat skin of lactating animal's dairy farm. Milking hygiene practices such as washing worker hands, rinsing udder and teats in sanitizing solution, then drying them, followed by effective germicidal teat dip could reduce pathogenic bacterial load on the udder skin. Further research should be carried out to explore other environmental risk factors responsible for intramammary infections within dairy herds.
Data Availability e datasets that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.