1. Introduction
There is no doubt that antibiotic resistance is one of the most pressing public health crises facing both the human and animal populations in the modern world. Yersinia spp., specifically Yersinia enterocolitica, is an example of an organism that develops resistance to a wide range of antibiotics ( 1 , 2 ). The extensive and indiscriminate application of antibiotics in veterinary medicine contributes to antibiotic resistance. Despite the inherent risks of bacterial contamination, it is still common practice to use these substances in surgical procedures aimed at sterilizing animals ( 3 , 4 ). The emergence of Yersinia's antibiotic-resistant strains has substantial consequences beyond geographic boundaries ( 5 ).
These resilient strains challenges the effectiveness of treatment methods and animal health practices. Addressing antibiotic resistance in Yersinia spp., and related pathogens is imperative due to animal and human health interdependence. The problem of bacterial resistance in veterinary medicine can further complicate public health concerns. It is vital to investigate Yersinia spp. susceptibility patterns to a wide range of antibiotics. A critical step towards safeguarding public health is understanding how bacteria respond to various antibiotics. The current study aims to determine which antibiotics are effective against Yersinia spp. when they adapt to antibiotics. This study includes insights into how veterinary medicine can make better decisions regarding treatment during sterilization procedures for better outcomes.
2. Materials and Methods
2.1. Sampling
Samples were collected from the surgical site before preparation for castration surgery in a sheep. Before starting the surgery, the site was thoroughly cleaned with alcohol, chlorhexidine, and povidone-iodine. The surgical site and suture location were swabbed following ceftriaxone administration and the completion of the procedure. After 24 hours of incubation of samples, following linear culture, crystal violet, iodine, and fuchsin stains were applied to determine the colony type.
As soon as bacteria were identified through Gram staining, they were cultured linearly within their specific media. To cultivate Gram-negative bacteria, including Yersinia, the EMB medium was used, along with Simmon's citrate, TCI, SIM, and MR-VP. A 24-hour incubation at 37°C was used to promote bacterial growth and stability after being cultured in their specialized media. Testing with Methyl Red was performed using an MR medium. A VP test was conducted using an MRVP medium divided into two parts. SIM medium, which is semisolid, is then used for further investigation ( 6 ).
2.2. Antibiotic susceptibility test
Gram-negative bacteria were cultured on Mueller-Hinton agar medium once removed from the refrigerator. By inserting six antibiotic disks, this study examined the susceptibility of several antibiotics, including nitrofurantoin, gentamicin, cefazolin, tetracycline, ceftriaxone, and ampicillin. The antibiotics chosen for this study were relevant to veterinary medicine, since veterinarians may use them postoperatively. Sterilization was achieved by disinfecting the disks with alcohol and incubating them in the culture medium. At 37°C, the plates were incubated for 24 hours. The inhibition zones were measured with a ruler after 24 hours of incubation. Antibiotic susceptibility testing was performed using standards established by the Clinical and Laboratory Standards Institute (CLSI) ( 7 , 8 ).
3. Results
3.1. Sampling culture findings
Among the colonies sampled before surgery, Gram-positive cocci, Gram-positive bacilli, and Gram-negative bacilli were found. After surgery, one Gram-negative colony was found in swab samples after staining and microscopic examination. An extensive series of biochemical tests determined the bacteria's identity. Testing the bacteria with the Methyl Red (MR) test determined that they did not produce acidic end products when they metabolized glucose. There was no evidence of acetoin production by the bacteria in Voges-Proskauer tests (VP). This suggests that enteric bacteria do not produce acetoin. During the Citrate utilization test, it was determined that the bacterium could utilize citrate alone as a carbon source. The bacteria showed positive results in a glucose fermentation test, indicating they can ferment glucose. A lack of these components prevented them from producing hydrogen sulfide (H2S), indole, or exhibiting motility. These tests contributed to identifying Yersinia spp. as the pathogen in the case under investigation.
3.2. Antibiogram
The inhibition zones for each antibiotic were measured to determine the bacterium's susceptibility to antibiotics (Figure 1).
Figure 1. The antibiotic disks were inserted into the media, allowing six different antibiotics to be tested for Yersinia resistance. The antibiotics utilized were ceftriaxone, nitrofurantoin, gentamicin, cefazolin, ampicillin and Tetracycline.
In addition, this bacterium displayed an extensive inhibitory zone (more than 17 mm), which qualified it as highly sensitive to nitrofurantoin. There was also evidence to indicate that the bacteria are highly susceptible to gentamicin, based on the fact that the zone diameter was 27 mm. Furthermore, tetracycline also showed an effect against the bacteria, displaying a zone diameter of 23 mm, indicating susceptibility. The zone diameter of the bacterium was only 11 mm, despite its resistance to ampicillin. As the zone diameters were 17 mm and 10 mm, it was evident that the bacterium was resistant to ceftriaxone and cefazolin (Table 1).
| Antibiotic | µAmount (g) | Zone Size (mm) | Sensitive | Intermediate | Resistance |
|---|---|---|---|---|---|
| Nitrofurantoin | 300 | 28 | 17≤ | 16-15 | 14≥ |
| Gentamicin | 10 | 27 | 15≤ | 13-14 | 12≥ |
| Tetracycline | 30 | 23 | 15≤ | 14-12 | 11≥ |
| Ceftriaxone | 30 | 17 | 23≤ | 22-20 | 19≥ |
| Ampicillin | 10 | 11 | 17≤ | 16-14 | 13≥ |
| Cefazolin | 30 | 10 | 23≤ | 22-20 | 19≥ |
4. Discussion
One of the critical pathogenic bacteria is Yersinia spp., a member of the Enterobacteriaceae family ( 9 ). There are several serotypes and biotypes of this bacterium. Among the factors contributing to its pathogenicity are its ability to grow at temperatures between 0 and 44°C and the diversity of its surface antigens. Yersinia spp. causes yersiniosis, a zoonotic disease affecting both humans and animals ( 10 ). Antibiotics are used in veterinary medicine as a treatments, preventatives, and even growth promoters, to manage infections and prevent infectious diseases ( 11 ).
However, resistance to antibiotics is on the rise, affecting both bacterial populations and different hosts. Evidence suggests that horizontal gene transfer and mobile genetic elements are responsible in the development of this resistance, which can reduce the effectiveness of antimicrobial agents in both humans and animals ( 12 ).
According to a study conducted in Bulgaria, Yersinia spp., isolated from pork were resistant to ampicillin, tetracycline, and nalidixic acid, yet sensitive to chloramphenicol and gentamicin ( 13 ). In Egypt, another study found the highest resistance to ampicillin, cefazolin, and amoxicillin/clavulanic acid among strains of Yersinia spp ( 14 ). Numerous studies have documented diverse antibiotic resistance patterns in Yersinia isolates ( 15 , 16 ). As a result of these patterns, it is imperative conducting localized susceptibility testing in specific regions ( 17 ). To successfully treat bacterial infections, it is crucial to choose effective antibiotics, particularly for highly pathogenic bacteria such as Yersinia.
Based on the results of the current study, the isolate of Yersinia spp. from castration surgical sites was very susceptible to nitrofurantoin and gentamicin. Due to their close relationship, these antibiotics may effectively treat and eradicate Yersinia spp. infections. The treatment of Yersinia spp. infection should not involve antibiotics like ampicillin, ceftriaxone, or cefazolin due to bacterial resistance to these drugs. Limitations of the present study include the small sample size and the limited number of control groups. In this study, we only examined bacteria isolated from castration surgery of male sheep, which may limit the generalizability of the results. To increase the accuracy and generalizability of the results, the number of samples in future studies should be increased, and the inclusion of additional control groups should be considered. Moreover, repeating the experiments under different conditions and using different laboratory methods can help improve the results' validity and accuracy. In addition, using animals from different breeds and conditions—especially in different geographical areas—can provide more information about antibiotic resistance patterns in various bacteria and help design more effective treatment strategies (Table 2).
| Challenge | Description | Suggested Improvement |
|---|---|---|
| Sample Size | The current study had a small sample size, limiting the ability to generalize findings. | Increase the sample size to ensure more robust and reliable results. |
| Control Group Limitations | Only a limited number of control groups were used, which may not reflect the broader spectrum of cases. | Add more control groups with diverse conditions to improve the validity of comparisons. |
| Replication of Experiments | The experiments were not repeated under different conditions, affecting the results' reproducibility. | Repeat experiments across different settings and methodologies to confirm consistency and reliability. |
| Animal Diversity | The study focused on a single breed of sheep, limiting the representation of different animal variations. | Include animals from different breeds and geographical regions to obtain a wider range of data on antibiotic resistance. |
| Geographical Variation | The study did not account for geographical differences in bacterial resistance patterns. | Perform studies in multiple regions to observe geographical trends in antibiotic resistance. |
| Data Interpretation | The limitations may influence the results of the testing methods and protocols used. | Use various testing methods to confirm findings and ensure comprehensive data interpretation. |
| Statistical Significance | The study might not have fully explored the statistical significance across different groups. | Perform deeper statistical analyses to explore the significance of observed differences in greater detail. |
As a revolutionary system, it is highly recommended that nanotechnology be considered for overcoming the challenge of drug resistance ( 18 ). Nanotechnology has attracted much attention due to its potential to improve disease diagnosis, treatment, and prevention, especially in human and veterinary medicine ( 19 ). Important applications of nanotechnology include designing nanoparticles for precise drug targeting, faster identification of pathogens, and the creation of intelligent drug delivery systems ( 20 ).
This technology can also help develop more accurate diagnostic tools and optimize treatments for drug-resistant diseases. Therefore, further research and investment in this area promise significant advances in various scientific and medical fields.
Various bacterial strains exhibit different antibiotic resistance profiles, so tailored treatment must be considered. According to the findings of this study, nitrofurantoin, Gentamicin, and Tetracycline may also be more effective than ampicillin, cefazolin, and ceftriaxone against Yersinia spp.
Acknowledgment
We sincerely thank Islamic Azad University, Babol Branch, Babol, Iran, for supporting us.
Authors' Contribution
Study concept and design: MB
Acquisition of data: ZA, NZ, MB
Analysis and interpretation of data: ZA, NZ, MB
Drafting of the manuscript: ZA, NZ, MB
Critical revision of the manuscript for important intellectual content:
Statistical analysis: ZA, NZ, MB
Administrative, technical, and material support: MB
Ethics
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. IR.IAU.BABOL.REC.1403.065.
Conflict of Interest
The authors declare no conflict of interest.
Funding
No funding was received.
Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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