|Year : 2022 | Volume
| Issue : 1 | Page : 16-20
Detection of Heteroresistant Vancomycin-Intermediate Staphylococcus aureus among Methicillin-Resistant Staphylococcus aureus
Bajarangi Lal Chaudhary1, Dakshina Bisht1, Sameer Singh Faujdar2
1 Department of Microbiology, Santosh Medical College and Hospital, Santosh Deemed to be University, Ghaziabad, Uttar Pradesh, India
2 Department of Microbiology, Maharishi Markandeshwar Medical College and Hospital, Maharishi Markandeshwar University, Solan, Himachal Pradesh, India
|Date of Submission||05-Dec-2021|
|Date of Decision||26-Dec-2021|
|Date of Acceptance||03-Jan-2022|
|Date of Web Publication||01-Mar-2022|
Department of Microbiology, Santosh Medical College and Hospital, Santosh Deemed to be University, Ghaziabad - 201 009, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: The testing of heteroresistance vancomycin-intermediate Staphylococcus aureus (hVISA) by the population analysis profile-area under the curve (PAP-AUC) is tedious and laborious. The goal of this study was to determine the prevalence of hVISA among methicillin-resistant S. aureus (MRSA) and compare two screening tests: brain heart infusion agar with vancomycin 4 mg/L (BHIV4) and macromethod E-test (MET) with PAP-AUC as the gold standard. Materials and Methods: Antibiotic sensitivity test and detection of MRSA were performed according to the CLSI guidelines. PAP-AUC, BHIV4, and MET were used to detect hVISA. Results: A total of 288 MRSA isolates were tested for hVISA, with 48 (16.66%) being detected by PAP-AUC. With PAP-AUC, BHIV4's sensitivity, specificity, positive predictive value, and negative predictive value were 70.83%, 96.66%, 80.95%, and 94.3%, respectively. The majority of hVISA isolates were found in the vancomycin minimum inhibitory concentration range 1–2 mg/L. Conclusions: hVISA was found to be 16.6% in this investigation. When compared to MET, BHIV4 had a higher sensitivity, and teicoplanin and linezolid were found to be the most effective drug against hVISA.
Keywords: Brain heart infusion agar with vancomycin 4 mg/L, heteroresistance vancomycin-intermediate Staphylococcus aureus, macromethod E-test, methicillin-resistant Staphylococcus aureus, population analysis profile-area under the curve, vancomycin
|How to cite this article:|
Chaudhary BL, Bisht D, Faujdar SS. Detection of Heteroresistant Vancomycin-Intermediate Staphylococcus aureus among Methicillin-Resistant Staphylococcus aureus. Asian J Pharm Res Health Care 2022;14:16-20
|How to cite this URL:|
Chaudhary BL, Bisht D, Faujdar SS. Detection of Heteroresistant Vancomycin-Intermediate Staphylococcus aureus among Methicillin-Resistant Staphylococcus aureus. Asian J Pharm Res Health Care [serial online] 2022 [cited 2022 Dec 6];14:16-20. Available from: http://www.ajprhc.com/text.asp?2022/14/1/16/338788
| Introduction|| |
Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically important bacterium in humans, causing infections in the eyes, cardiovascular system, skin and soft tissues, respiratory tract, and circulatory system. Since the emergence of vancomycin resistance in Enterococci in 1988, glycopeptides have been indicated as therapeutic agents for treating MRSA, which is often multiresistant. For the past four decades, there has been no report of vancomycin resistance in S. aureus., In 1996, the first vancomycin-intermediate S. aureus (VISA) isolate was reported. The first heteroresistant VISA (hVISA) and vancomycin-resistant S. aureus isolates appeared shortly after. The occurrence of a resistant subpopulation in hVISA isolates which occurs at a rate of 1 in 105 organisms indicates an intermediate stage between vancomycin susceptible S. aureus (VSSA) and VISA isolates. The hVISA and VISA isolates have subsequently been recognized worldwide; however, because testing procedures are not standardized, it is challenging to identify the exact prevalence of hVISA. The population analysis profiling-area under the curve method (PAP-AUC) is the current gold standard method. However, it is expensive, time-consuming, and labor-intensive. Other procedures such as the macro method E-test (MET) and screening plates have been reported but none of them compare to the PAP-AUC method in terms of sensitivity and specificity. A number of alternative methods for detecting the heteroresistant phenotype have been tested with variable degrees of success and no single standardized clinical laboratory procedure has yet been created. As a result, this study was done to determine the feasibility of diagnosing hVISA by screening BHI agars enriched with 4 mg/L vancomycin and macro E-test.
| Materials and Methods|| |
This research was conducted in the Department of Microbiology, Santosh Medical College, Ghaziabad, in collaboration with Mayo Institute of Medical Sciences, Barabanki, Uttar Pradesh, India. The Institutional Ethics Committee granted their approval with reference number-SU/2018/528. All S. aureus isolates from the microbiology laboratory were identified using standard methods and further tested for MRSA screening by cefoxitin disc diffusion method and antibiotic susceptibility testing was performed according to the CLSI guidelines. A total of 288 MRSA-positive clinical isolates were examined further for their hVISA status.,,
Heteroresistance vancomycin-intermediate Staphylococcus aureus detection by brain heart infusion agar with vancomycin 4 mg/L method
All isolates were inoculated with 4 mg/l vancomycin (HiMedia, Mumbai) on brain heart infusion agar. On brain heart infusion agar with vancomycin 4 mg/L (BHIV4), a spot inoculation of 10 μl of a 0.5 McFarland bacterial solution was performed. Growth after 48 h was considered to be hVISA. For each plate, ATCC S. aureus 25923 was used as a negative control, while ATCC-700698, Mu3 strain of hVISA was utilized as a positive control.
Heteroresistance vancomycin-intermediate Staphylococcus aureus detection by macromethod E-test
The isolates were prepared as 2.0 McFarland saline suspensions and swabbed onto the BHIA plates and then left to dry, vancomycin E-test strips were applied on each strain separately. The MICs were determined after a 48-h incubation period at 37°C. The MET used the presence of one or more subpopulation colonies with MIC 4 mg/L as a criteria for finding hVISA.
Population analysis profile-area under the curve method
For the testing of hVISA by PAP-AUC, the few colonies of isolates were inoculated on BHI broth and then incubated at 37°C. After incubation, the bacteria were Log dilutions prepared ranging from (10−1–10−6). 100 μl of 10−3 – 10−6 dilutions were lawn cultured onto a set of BHI agar containing vancomycin at concentrations ranging from 0 to 8 mg/L. The plates were incubated for 48 h at 37°C. Using colony-forming unit/ml values, the AUC was determined by using GraphPad Prism software. The hVISA (ATCC-700698, Mu3) was used as positive control and S. aureus (ATCC-29213) was used as negative control strains. If the ratio of AUC of test to the AUC of control was found to be between ≥0.9 and ≤1.3 considered hVISA.
| Results|| |
A total of 655 S. aureus were isolated from various clinical samples. Of these, 288 isolates were MRSA through screening by cefoxitin disc diffusion method and were further tested for their vancomycin heteroresistant status. PAP-AUC test demonstrated that 48 (16.66%) MRSA strains had hVISA status while 240 (83.34%) strains were negative, whereas MET showed that 44 (15.27%) MRSA isolates were positive for hVISA status and 244 (84.73%) were negative as shown in [Table 1]. Among 48 hVISA strains, most of the strains (39.58%) showed 1.5 mg/L MIC value of vancomycin followed by 35.42% strains showed 3 mg/L and 25% strains showed 2 mg/L MIC value, as shown in [Figure 1.
|Figure 1: Distribution of MIC value of heteroresistance vancomycin-intermediate Staphylococcus aureus isolates|
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|Table 1: Heteroresistance vancomycin-intermediate Staphylococcus aureus detection in methicillin-resistant Staphylococcus aureus strains by macro E-test and population analysis profile-area under the curve test|
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BHIV4 test revealed that only 42 (14.58%) MRSA strains were able to grow on BHI agar having 4 mg/L vancomycin and 246 (85.42%) strains could not grow, as shown in [Table 2].
|Table 2: Heteroresistance vancomycin-intermediate Staphylococcus aureus detection in methicillin-resistant Staphylococcus aureus strains by brain heart infusion agar with vancomycin 4 mg/L test and population analysis profile-area under the curve test|
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PAP-AUC test was considered a standard test for hVISA detection and further results were compared with MET and BHIV4 method, and performance of these two tests was detected in terms of sensitivity, specificity, positive predictive value, and negative predictive value as described in [Table 3].
|Table 3: Performance of heteroresistance vancomycin-intermediate Staphylococcus aureus by screening methods|
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The PAP-AUC ratios (ratio of the AUC of the isolate to the AUC of Mu3) ranged from 0.9 to 1.29 with a mode of 1.1 and 1.2 for the 48 hVISA MRSA isolates as shown in [Figure 2].
|Figure 2: Population analysis profile-area under the curve ratio of heteroresistance vancomycin-intermediate Staphylococcus aureus (n = 48)|
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The antibiotic resistance patterns of hVISA isolates showed that erythromycin and azithromycin show the highest rate of resistance of 82.6% and 80.42%, respectively, whereas teicoplanin and linezolid show low rate of resistance of 10.4% and 10.42%, respectively, as described in [Figure 3].
|Figure 3: Antibiotic resistance among heteroresistance vancomycin-intermediate Staphylococcus aureus (n = 48)|
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| Discussion|| |
Although the prevalence of hVISA among MRSA in many hospitals is still unknown, it is critical for infection control management. Because the Stoke disk diffusion approach fails to detect the hVISA, a standard detection method is necessary. The broth dilution test, agar incorporation test and the traditional E-test are other procedures for determining hVISA MICs. The effectiveness of the tests is still debatable. The lack of established diagnostic methodologies, as well as the varying prevalence of hVISA as described in research, adds to the problem. PAP is the most widely used method for identifying hVISA, but it is unlikely to be used in clinical laboratories.
The occurrence of hVISA has been reported in about 6.81% in Asian countries and 5.6% in Europe and America. Whereas Indian authors, Song et al., Iyer and Hittinahalli and Chaudhari et al., Singh et al., and Amberpet et al. documented prevalence of 6.3%, 2%, 6.9%, 5.8%, and 12.4%, respectively, at an average of 6.8% among the total of 1188 MRSA isolates. In the present study, 288 MRSA isolates were checked for the hVISA and it was found to be 16.6%. Multiple drug resistance has become a major concern in the situation of hVISA infection. The current study reveals that erythromycin and azithromycin had shown the highest rate of resistance (82.6% and 80.42%, respectively), whereas teicoplanin and linezolid had shown the lowest rates of resistance (10.4% and 10.42%, respectively). On the other hand, a study done by Shang-Yi Lin et al. documented that ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and tetracycline showed high resistance rates and daptomycin, linezolid, and tigecycline showed very less resistance rates against hVISA isolates.
In this investigation, 48 MRSA isolates were hVISA using the PAP-AUC method and AUC values range from a ratio of 0.9–1.3 with the mode of 1.1 and 1.2, whereas Satola et al. found the ratio ranged from 0.57 to 1.48, with a mode of 0.74. In our investigation, the majority of the strains (39.58%) had a vancomycin MIC of 1.5 mg/L, whereas Amberpet et al. found that the majority of the isolates had vancomycin MIC 1-2 mg/L which was quite similar to the current study. The current study's BHIV4 results were quite comparable to those of Amberpet et al., however, in the different study by Ridere et al., good BHIV3 results were obtained, which validated the MET approach. Because the PAP-AUC test has been regarded as the gold standard for detecting hVISA, it is now the appropriate time to detect hVISA in diagnostic laboratories on a routine basis. Similarly, we should promote the use of alternative screening tests to detect hVISA, such as the BHIV4, MET, and glycopeptide resistance detection tests.
| Conclusions|| |
Early detection of hVISA status in MRSA could help to prevent treatment failure and MRSA-related infections. The PAP-AUC technique revealed a 16.6% occurrence of hVISA in the current study. In comparison to the MET test, the BHIV4 technique was found to be more effective in detecting hVISA. Among hVISA isolates, the PAP-AUC ratios had a mode of 1.1. The antibiotics teicoplanin and linezolid are the medications of choice for the treatment of hVISA.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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