European Journal of Cardiovascular Medicine

The establishment and spread of antibiotic resistance among bacterial pathogens constitute a substantial hazard to the health of people all over the world. This poses a challenge to the efficacy of antimicrobial treatment and makes the management of infectious illnesses more difficult. Staphylococcus aureus, which is a primary source of infections that are acquired in the community as well as those that are connected with healthcare, has shown amazing flexibility by developing mechanisms that are resistant to different medications.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen that causes infections in both hospital and community settings. Vancomycin is the first line of defence against potentially life-threatening MRSA infections.  Elevated vancomycin MICs can lead to MRSA resistance and treatment failure.

Vancomycin is a crucial antibiotic that is often regarded as a last option for the treatment of severe infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcus aureus strains that exhibit resistance to vancomycin are a particular reason for worry. It is possible for this resistance to appear in a variety of forms, such as vancomycin-resistant Staphylococcus aureus (VRSA) and vancomycin-intermediate Staphylococcus aureus (VISA), which presents considerable hurdles to clinical care[1].

Influenced by variables such as antibiotic use, infection control strategies, and bacterial genetic diversity, the frequency of vancomycin-resistant Staphylococcus aureus (VSSA), vancomycin-resistant Staphylococcus aureus (VRSA), and vancomycin-sensitive Staphylococcus aureus (VSSA) strains vary regionally and temporally[2].In order to guide empirical therapy, optimize treatment regimens, and implement successful infection control methods, it is essential to have a solid understanding of the antimicrobial sensitivity patterns of these strains. A further benefit of monitoring resistance trends is that it offers significant insights into the dynamics of the evolution of bacterial resistance and contributes to the development of innovative treatment methods.

Antibiotic Resistance in Staphylococcus aureus:

Antibiotic resistance is a serious problem in the field of public health and Staphylococcus aureus is a prime example of this dilemma because of its extraordinary capacity to acquire resistance to a variety of antimicrobial drugs. Because it is a versatile bacterium that may be found in a variety of habitats, including the skin and mucous membranes of humans. Staphylococcus aureus is a bacteria that is often responsible for infections. Boils and abscesses are examples of mild skin and soft tissue infections. On the other hand, bloodstream infections and pneumonia are examples of serious and potentially life-threatening circumstances that may be caused by these illnesses[3,4]. As a result of the appearance of methicillin-resistant Staphylococcus aureus (MRSA) strains, the difficulty of treating infections caused by Staphylococcus aureus has increased. In the past, beta-lactam antibiotics, such as methicillin and various penicillins, were considered to be effective treatments for staphylococcal infections. However, MRSA strains have developed resistance to these prescription drugs. In order to effectively treat MRSA infections, other antibiotics, such as vancomycin, have emerged as an essential therapeutic option. However, the increased occurrence of vancomycin-resistant Staphylococcus aureus (VRSA) strains has further confounded treatment choices, underlining the critical need for effective measures to address antibiotic resistance in this bacterium.

       Vancomycin Resistance in Staphylococcus aureus:

A significant obstacle that must be overcome in clinical settings is the presence of vancomycin resistance in Staphylococcus aureus (VRSA). Vancomycin, which is a glycopeptide antibiotic, has been considered for a long time to be the last line of defense against severe infections that are caused by methicillin-resistant Staphylococcus aureus (MRSA). On the other hand, the effectiveness of this essential antibiotic is being undermined by the appearance and proliferation of VRSA strains. In most cases, VRSA strains develop resistance by acquired genetic pathways, which often include the acquisition of vanA or vanB genes. Because these genes generate altered cell wall precursors, the binding affinity of vancomycin to its target site is decreased. As a result, the antibiotic is unable to effectively suppress the development of bacteria. Furthermore, vancomycin-intermediate Staphylococcus aureus (VISA) strains have been discovered. These bacteria have a decreased sensitivity to vancomycin, but they do not fulfill the requirements for complete resistance. The introduction of VRSA and VISA strains makes treatment plans more difficult to implement since these strains need alternate therapeutic procedures and raise worries about the possibility of resistance spreading. Therefore, in order to effectively manage the rising danger of vancomycin resistance in Staphylococcus aureus infections, it is essential to have good monitoring, infection control measures, and the development of novel treatment modalities[5].

       Significance of Antimicrobial Sensitivity Patterns:

It is impossible to emphasize the relevance of antibiotic sensitivity patterns in Staphylococcus aureus, especially in the context of vancomycin-resistantStaphylococcus aureus (VRSA), vancomycin-intermediateStaphylococcus aureus (VISA), and other strains of Staphylococcus aureus within the framework of clinical practice and attempts to manage infections. To begin, these sensitivity patterns serve as vital recommendations for empirical therapy. They enable doctors to choose the antibiotic regimen that is the most suited for first treatment, even before the findings of precise pathogen identification and susceptibility testing are known. Particularly important in the treatment of serious infections, when the prompt administration of antibiotics that are effective may have a substantial influence on the outcomes for patients, this is very essential. Furthermore, doctors are able to customize treatment regimens to specific patients based on their antimicrobial sensitivities when they have a thorough grasp of the susceptibility profiles of VRSA, VISA, and VSSA strains to a panel of antibiotics. The use of this individualized method helps to maximize the effectiveness of therapy while simultaneously reducing the likelihood of unpleasant effects caused by antibiotics and the development of further resistance. Furthermore, the development of resistance mechanisms and the spread of resistant strains within healthcare settings and communities may be better understood by the ongoing monitoring of resistance trends over time. This gives useful information. Early diagnosis of developing resistance patterns enables prompt actions, such as revisions in antibiotic prescription practices, deployment of infection control measures, and focused antimicrobial stewardship activities aimed at conserving the efficacy of current medicines. These interventions are taken in order to prevent the spread of antibiotic-resistant bacteria[6].

The purpose of this research was to evaluate the antimicrobial sensitivity patterns of VRSA, VISA, and VSSA that have been isolated from a variety of clinical specimens respectively. We want to understand the range of resistance characteristics shown by these bacteria by conducting an analysis of a comprehensive collection of specimens derived from a variety of patient groups and clinical settings. Additionally, we had evaluate the susceptibility of these strains to alternative antimicrobial drugs. Our knowledge of the changing landscape of Staphylococcus aureus resistance was predicted to be improved as a result of this research, contribute to the sensible use of antibiotics in clinical practice will be useful.

Research Design:

       This study employed a cross-sectional observational design to assess the antimicrobial sensitivity patterns among vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-intermediate Staphylococcus aureus (VISA), and vancomycin-sensitive Staphylococcus aureus (VSSA) isolated from various clinical specimens.

       Data Collection and Procedure:

       Place of Study: The study was conducted in the Department of Microbiology, Government Medical College, Kota, Rajasthan, India.

       Duration of Study: Data collection took place over a period of three years, starting from September 7, 2019, to September 6, 2022, following approval from the Departmental Research Committee (DRC).

       The Ethical Letter: The Ethical clearance was duly obtained from the Institutional Medical College of GMC, Kota.

        Procedure

The vancomycin minimum inhibitory concentrations (MICs) of the isolates were determined using bothagar dilution method and E-test strip method. Isolates were categorized as VRSA (MIC ≥ 16 μg/ml), VISA (MIC = 4-8 μg/ml), or VSSA (MIC ≤ 2 μg/ml) based on their vancomycin MICs.

Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method for a panel of antibiotics. The results were interpreted according to CLSI guidelines and isolates were classified as susceptible, intermediate, or resistant.

       Statistical Analysis

Data were analyzed using appropriate statistical methods, including descriptive statistics to summarize the antimicrobial sensitivity patterns among VRSA, VISA, and VSSA isolates. Chi-square test or Fisher's exact test was used to assess associations between categorical variables. Statistical significance was set at p < 0.05. All analyses were performed using statistical software (e.g., SPSS, SAS).

In the present study a total of 185 Methicillin resistantStaphylococcus aureus isolates were detected from different clinical specimens.

Table 1: Determination of VRSA, VISA and VSSA among MRSA isolates by Agar dilution method and E-test strip method on the basis of MIC values.

MRSA isolates were subjected to MIC determination by the agar dilution method and E-test strip method. Out of the 185 MRSA isolates 02 isolates were identified as VRSA and 03 isolates were identified as VISA by both methods.

Fig. 1: Determination of VRSA, VISA and VSSA isolates among MRSA by Agar dilution method and E-test strip method on the basis of MIC values.

Table 2: Age and sex wise distribution of VRSA, VISA and VSSA isolates among MRSA.

M: Male   F: Female

Out of  02 (1.08%) VRSA isolates  of 185 (100%) MRSA isolates, 01 (1.02%) isolate was from male patient out of 98 (52.97%) MRSA male patients and 01 (1.15%) was from female patient out of 87 (47.03%
) MRSA female patient. The 01 VRSA isolate from male patients, belonged to (41-50) age group. The 01 VRSA isolate from female patients, belonged to (61-70) age group.

Among 03 (1.62%) VISA isolates out of 185 (100%) MRSA isolates, 02 (2.30%) isolates were from female patients out of 87 (47.03%) female MRSA isolates and 01 (1.02%) isolate was from male patient out of 98 (52.97%) male MRSA isolates. Among 02 (2.30%) VISA isolates from female patients, 01 belonged to (00-10) age group and 01 was from (61-70) age group. 01 (1.02%) VISA isolate was from male patients belonged to 71-80 age group.

Fig. 2: Age and Sex wise separation of VRSA, VISA and VSSA isolates among MRSA.

Table 3: Distribution of VRSA, VISA and VSSA isolates obtained from various clinical samples among MRSA.

χ2 : Chi square test   Df: Degree of freedom      NS: non-significant (p>0.05 at 95% CI(Confidence Interval)

VRSA isolates:

          All 02 (1.08%) VRSA isolates were from pus sample.

VISA isolates:

In the current study out of the 03 (1.62%) VISA isolates, 02 were from pus and 01 was observed  from blood. The results were statistically insignificant (p>0.05).

Fig 3: Distribution of VRSA, VISA and VSSA isolates obtained from various clinical samples among MRSA.

Table 4: Antimicrobial susceptibility pattern among VRSA, VISA and VSSA isolates.

S: Sensitive     I: Intermediate          R:Resistant        χ2 : Chi square test       df: Degree of freedom           *: Significant (p<0.05 at 95% CI (Confidence Interval)     **: Significant (p<0.01 at 99% CI (Confidence Interval)

VRSA, VISA and VSSA isolates shows variable antimicrobial sensitivity pattern against antimicrobial agents of different classes. Out of 2 VRSA strains, 2 (100%) were sensitive to both Teicoplanin and Linezolid, followed by Cefepime 1 (50%), Tetracyclin 1 (50%), Amikacin 00 (0%), Cloxacillin 00 (0%), Levofloxacin 00 (0%), Clindamycin 00 (0%), Cefazolin 00 (0%), Erythromycin 00 (0%), Cefoperazone 00 (0%), Cotrimoxazole 00 (0%) and Nitrofurantoin 00 (0%).Out of 3 VISA strains, 3 (100%) were sensitive to Linezolid, followed by Teicoplanin 2 (66.67%), Cefepime 2 (66.67%), Tetracyclin 1 (33.33%), Amikacin 01 (33.33%), Levofloxacin 01 (33.33%), Clindamycin 01 (33.33%), Cefoperazone 01 (33.33%), Quinopristin/Dalfopristin 01 (33.33%), Cloxacillin 00 (0%),  Cefazolin 00 (0%), Erythromycin 00 (0%), Cotrimoxazole 00 (0%) and Nitrofurantoin 00 (0%).

Out of 180 VSSA strains, 180 (100%) were sensitive to  Vancomycin followed by Linezolid 173 (96.11%), Teicoplanin 162 (90%), Cefepime 146 (81.11%), Amikacin 141 (78.33%), Tetracyclin 133 (73.89%), Levofloxacin 122 (67.78%), Clindamycin 117 (65%), Cefazolin 111 (61.67%), Erythromycin 104 (57.78%), Quinopristin/Dalfopristin 96 (53.33%), Cefoperazone 91 (50.56%), Cloxacillin 82 (45.56%), Cotrimoxazole 48 (26.67%)  and Nitrofurantoin 24 (88.89%) out of 27 urine isolates. Statistical significance was obtained only in cases of Amikacin (p<0.01), Cefazolin (p<0.05),

Cefepime (p<0.05), Erythromycin (p<0.01) and Nitrofurantoin (p<0.01

Fig.4: Antimicrobial susceptibility pattern among VRSA, VISA and VSSA isolates

Table 5: Comparative determination of VRSA, VISA and VSSA isolates by Agar dilution method and E-test strip method on the basis of MIC values.

By agar dilution method and E-test strip method 2 (1.08%) VRSA and 3 (1.62%) of VISA isolates were detected(table 5). This result was comparable to the study byThati V et al who documented 1.9% of VRSA strains and 4.46% of VISA strains. In the study of Osman MM et al, there was no isolation of any VRSA stains and the overall prevalence of VISA was found to be 12%. They were mostly observed in the patients having underlying conditions such as long-term hospitalization, serious disease and immune-suppressive therapy.

Study of Maharjan M et al detected 11.11% of VRSA strains. Of these isolates detected, 40% of the isolates demonstrated Van A gene only while rest demonstrated both Van A and Van B genes (A or B) in 40% of the VRSA isolates while the remaining isolates possessed other van genes.    In the study of Nepal N et al, all 35 MRSA isolates contained mecA gene while 2 of them also possessed van B gene, both of which showed intermediate resistance vancomycin of MIC 8 ug/ml. None of the isolates were found to have van B gene [10].

Table 6: Comparison of age and gender-based prevalence of VRSA isolates with other studies

The VRSA isolate in male patients was found from the age group of 41-50 years while in case of female patients, it was observed from the age group of 61-70 years. Among 3 VISA isolates, 1 isolate obtained in male patients belonged to the age groups of 71-80 years and 2 isolates obtained in female patients belonged to the age groups of 0-10 years and 61-70 years each (table 6). There was no significant difference in the incidence of these isolates with age and among male and female gender.

The study by Sarrafzadeh F et al, in their study, examined 250 positive specimens of staph aureus for vancomycin resistance. They reported 15 specimens from males and 8 specimens from females to be vancomycin resistant indicating high rate of resistance in female patients. Most of the VRSA were obtained from the patients of 19-64 years age group. Likewise, in the study of Tefera Set al, most of the VRSA isolates were detected in the age group of 31-45 years. From both male and female patients 5 isolates each of VRSA were obtained. There was no significant correlation between the rate of resistance and the age of the patient which in accordance to the present study.

Table 7: Comparison of prevalence of VRSA isolates in various clinical specimen with other studies

In this study, all the 2 (40%) VRSA isolates were obtained from the pus sample. Likewise, of 3 VISA isolates, 2 (40%) were obtained from pus and 1(20%) was from the blood sample. In the study of Nepal N et al, 4 VISA isolates were detected of which 3 (75%) were obtained from pus sample and 1 (25%) from wound swab. 2 VISA isolates possessed van B gene (one isolate each from pus sample and wound swab) (table 7). Likewise, in the study of Al-Ghareeb KAet al, of total 12 VRSA isolates, 3 (25%) isolates each were detected from pus and sputum samples. In contrast, Sarrafzadeh F et al showed the majority of VRSA isolates to be from CSF samples (14/60.86%).

Table 8: Antimicrobial susceptibility pattern for Amikacin among VRSA, VISA and VSSA isolates. 

In the present study 33.33% of VISA and 78.33% of VSSA strains were sensitive to amikacin. All VRSA strains were resistant to amikacin. Comparative evaluation of sensitivity pattern in various studies is shown in table 8. In the study of Mandal M et al, 75% of VRSA, 90.91% of VISA and 83.87% of VSSA isolates were sensitive to amikacin. The sensitivity of VRSA isolates to amikacin was higher than that observed in the present study.

Table 9: Antimicrobial susceptibility pattern for Clindamycin among VRAS, VISA and VSSA isolates. 

In the present study 33.33% of VISA and 65% of VSSA strains were sensitive to clindamycin. All VRSA strains were resistant to clindamycin. Comparative evaluation of sensitivity pattern in various studies is shown in table 9. In the study of Mandal M et al, 75% of VRSA, 72.73% of VISA and 56.99% of VSSA isolates were sensitive to clindamycin. The sensitivity of VRSA isolates to clindamycin was higher than that observed in the present study.

Table 10: Antimicrobial susceptibility pattern for Teicoplanin among VRSA, VISA and VSSA isolates. 

In the present study 66.67% of VISA and 90% of VSSA and all VRSA strains were sensitive to teicoplanin. Comparative evaluation of sensitivity pattern in various studies is shown in table 10. In the study of Mandal M et al, all VRSA, 18.18% of VISA and 87.1% of VSSA isolates were sensitive to teicoplanin. The sensitivity of VRSA isolates to clindamycin was similar to that observed in the present study.

Table 11: Antimicrobial susceptibility pattern for Linozolid among VRSA, VISA and VSSA isolates. 

In the present study 100% of VRSA, 100% of VISA and 96.11% of VSSA strains were sensitive to linozolid. Comparative evaluation of sensitivity pattern in various studies is shown in table 11. In the study of Mandal M et al, 50% of VRSA, 100% of VISA and 100% of VSSA isolates were sensitive to linozolid. The sensitivity of VRSA isolates to linozolid was higher in the present study.

All the VRSA and VISA strains were resistant to cefazoline, cotrimoxazole, cloxacillin, erythromycin, vancomycin and nitrofurantoin while 61.67%, 26.67%, 45.56%, 57.78%, 100% and 88.89% of VSSA strains were sensitive respectively. All the VRSA strains were resistant to cefoperazone, levofloxacin, quinopristin/dalfopristin while 33.33% each of VISA and 50.56%, 67.78% and 52.33% of VSSA strains were sensitive to them. In case of cefepime, 50% of VRSA, 66.7% of VISA and 81.11% of VSSA were sensitive while in case of tetracycline, 50% of VRSA, 33.33% of VISA and 73.89% of VSSA strains were sensitive.

As per CLSI guidelines, the Staphylococcus aureus strains with MIC of ≤2 µg/mlwas considered as VSSA, VISA strains with MIC of 4-8 µg/ml and VRSA strains with MIC of ≥16 µg/ml. The emergence of VISA is due to the accumulation of gradual mutations of VISA associated genes [15], however, the mechanism that links diverse mec genes with VISA phenotypes remains yet unclear and needs further investigations.

It is possible to gain valuable insights into the ever-changing landscape of antimicrobial resistance by conducting an analysis of antimicrobial sensitivity patterns among vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-intermediate Staphylococcus aureus (VISA), and vancomycin-sensitive Staphylococcus aureus (VSSA) isolates

In the present study linezolid and teicoplanin were found to be most active against the VRSA strains as the VRSA isolates were sensitive to these antibiotics. Vancomycin and teicoplanin were introduced as a treatment choice for MRSA infections. However, Staphylococcus aureus with intermediate resistance to vancomycin (VISA) and complete resistance to vancomycin (VRSA) have abruptly emerged in past few decades thus making it a matter of public health concern.It is crucial to detect MRSA rapidly and accurately so that proper antimicrobial therapy can be administered and the spread of these strains is controlled [16].

The results highlight the issues that are faced by VRSA and VISA strains, which demonstrate variable degrees of resistance to antibiotics that are widely utilized. The decreased susceptibility of MRSA to vancomycin highlights the need for alternative treatment methods. Staff and hospitalized patients can significantly reduce MRSA spread by maintaining proper hygiene, and the implementation of well-defined antibiotic stewardship programs at both regional and national levels can further contribute to this effort [17,18].

Our study concluded that linezolid and teicoplanin were found to be most effective against the VRSA and VISA strains. These antibiotics can be utilized asthe drugof choice against VRSA, VISA and MRSA infections.The significant resistance rates that were reported among VRSA and VISA isolates highlight the need of establishing tight infection control measures and antimicrobial stewardship programs in order to reduce the spread of resistance and maintain the effectiveness of antimicrobials that are already in use. Furthermore, the discovery of antimicrobial susceptibility patterns provides information that is useful for empirical treatment and makes it easier to pick suitable antibiotic regimens that are suited to the specific requirements of particular patients.

       To fight antimicrobial resistance in Staphylococcus aureus infections, it is vital to maintain monitoring and research activities in order to track resistance trends, discover developing resistance mechanisms, and create innovative treatment options. Continued surveillance and research efforts are needed.

Declarations:

Conflicts of interest: There is no any conflict of interest associated with this study

Consent to participate: We have consent to participate.

Consent for publication: We have consent for the publication of this paper.

       Authors' contributions: All the authors equally contributed the work.

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