Presence of multiple van genes among glycopeptide non-susceptible Staphylococcus aureus exhibiting in vitro MIC creep phenomenon: A study from north-east India

Material & Methods

This study was undertaken at the department of Microbiology, Silchar Medical College and Hospital, Silchar, Assam, India from September 2018 to August 2022 after obtaining the protocol approval from the Institutional Ethics Committee.

Results

Among 845 isolates, 246 were confirmed as S. aureus based on the VITEK^® 2 compact instrument. Of the 69 were screened as glycopeptide non-susceptible. While conducting an MIC study, it was observed that 37 isolates were in the intermediate and 13 were in the resistant range against vancomycin (Table I). Towards teicoplanin, 39 isolates were within the intermediate range (16 μg/ml), and 30 were resistant (Table I). PCR assay confirmed a total of 12 isolates were harbouring vanA, followed by vanD (n=8) and vanB (n=7). Isolates with an MIC range of 2-16 μg/ml for vancomycin and 8-16 μg/ml for teicoplanin were selected for MIC creep analysis. Eight isolates were chosen for the study, covering each MIC range. While analyzing vancomycin MIC creep, four showed a significant increase in MIC (Fig. 1; Table II). In contrast, no creep phenomenon was observed for the rest of the four isolates. In the case of teicoplanin, seven isolates showed the MIC creep phenomenon (Fig. 2; Table III). While performing revertant analysis of all the isolates that showed MIC creep phenomenon for vancomycin and teicoplanin, it was observed that the isolates reverted to their initial MIC mostly in between 2-3 wk for vancomycin. The same phenomenon was observed for teicoplanin in 1-2 wk (Fig. 3 and 4; Table IV and V). However, when isolates were subjected to serial passages for 30 consecutive days without any antibiotic pressure, the MIC of vancomycin and teicoplanin came down to 1-0.25 μg/ml of the antibiotic.

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Fig. 1.

Four Staphylococcus aureus isolates (isolates 1, 4, 6, 7) showing the minimum inhibitory concentration (MIC) creep phenomenon against vancomycin.

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Fig. 2.

Seven S. aureus isolates (isolates 1, 3, 4, 5, 6, 7, 8) showing the minimum inhibitory concentration (MIC) creep phenomenon against teicoplanin.

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Fig. 3.

Revertant analysis of four S. aureus isolates that showed MIC creep against vancomycin have reverted back to their respective original MIC on withdrawn of vancomycin pressure.

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Fig. 4.

Revertant analysis of seven S. aureus isolates that showed MIC creep against teicoplanin have reverted back to their respective original MIC on withdrawn of teicoplanin pressure.

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Discussion

Vancomycin, the most commonly used glycopeptide antibiotic, is one of the empiric treatment options for MRSA infections¹⁹. Glycopeptide non-susceptibility among S. aureus is a serious concern that restricts treatment options within clinical settings. In the current study, glycopeptide non-susceptibility was found in eight per cent of the isolates, which agrees with a previous study where nine per cent of hVISA was recorded²⁰. A study conducted in Italy 2012 by Tascini et al²¹ revealed that out of 91 clinical isolates of S. aureus, 10 (9.9%) were resistant to teicoplanin, and 5 (5.5%) were resistant to vancomycin. Shariati et al²² conducted a meta-analysis and systematic review on the prevalence of hVISA/VISA/VRSA. After data analysis from 82 studies, it was found that the overall prevalence of VRSA was 1.5 per cent, VISA was 1.7 per cent, and hVISA was 4.6 per cent, and after 2010, it increased to 2.4, 4.3, and 5.3 per cent, respectively²². In India presence of vanA was reported from S. aureus²³. Where as the current study reported the presence of multiple van genes. In this study, vancomycin and teicoplanin MIC creep was observed in vitro condition that supports prolonged exposure to vancomycin increases the MIC of susceptible isolates. Furthermore 69 out of 246 S. aureus isolates were glycopeptide non-susceptible, of which 37 were of the VISA phenotype. This imposes the risk of adverse clinical outcomes if not detected early. A variable MIC range of isolates towards vancomycin and teicoplanin was observed in the study. Total 29 isolates in the susceptible MIC range against vancomycin have a potential risk of attaining hVISA phenotype in the future, severely compromising the glycopeptide treatment option. A study conducted in China showed vancomycin MIC creep in S. aureus isolates throughout the five yr study period²⁴. There are a few contemporary reports from India where hVISA was reported within diabetic and non-diabetic individuals. They observed an occurrence rate of 6.4 per cent hVISA within MRSA isolates²⁵. Another Indian study observed reduced vancomycin susceptibility (11.6%) in S. aureus²⁶. Similarly, another report from south India observed 12 per cent hVISA within diverse amino acid substitution in tcaRAB, vraSR and graSR genes²⁷. Recent studies from abroad also reported the presence of VISA/VRSA among MRSA strains²⁸. A study from Saudi Arabia²⁹ has reported vancomycin MIC creep in S. aureus over three years, which declined subsequently over the next three years’ time. VISA/hVISA phenotypes are reported to be associated with a mutation on vraSR and graSR, two-component regulation systems. It was observed that the constructs (mutants) demonstrated a remarkable increase in vancomycin MIC³⁰. Thus, the in vitro gradual increase in vancomycin MIC in the current study might have a link with mutations in regulatory regions. A study from the USA³¹ highlighted the MIC creep phenomenon over some time towards vancomycin. Recently, a study from India showed progressive MIC creep towards teicoplanin³². In the present study, all the isolates that showed MIC creep against vancomycin and teicoplanin reverted to a susceptible MIC range when antibiotic stress was withdrawn for 30 days. However, no study could be found to compare with the findings of the present study. Yeh et al³³ in 2012 showed increased vancomycin usage, resulting in vancomycin MIC creep in MRSA. It was also reported that vancomycin usage in 30 days before the isolation of a S. aureus culture had a higher MIC. However, this increase could not be correlated with higher mortality³³. The findings of this study will augment global knowledge of antimicrobial resistance. Our observations on low vancomycin MIC (4 µg/ml) of vanD harboring isolates and moderate to high vancomycin MIC (8-64 µg/ml) of vanA and vanB harboring isolates could underscore how these resistance determinants confer different phenotypes. In the present study, the in vitro MIC creep phenomenon was observed in laboratory conditions, advocating studies to be undertaken over two to three yr to understand and correlate with glycopeptide usage and any increase in MIC. The findings of this study emphasize the need for a local epidemiological cut-off point for screening resistant pathogens. This warrants designing future diagnostics that can effectively detect heterogeneous resistant populations of bacteria. The current study could predict how these glycopeptide nonsusceptible isolates can attain a higher inhibitory concentration within the patient population when initiating glycopeptide treatment. This also highlights the adoption of testing facilities for glycopeptide non-susceptibility within routine microbiology laboratories. However, the information of the current investigation is restricted to in vitro analysis only.

Overall, the study found the prevalence of multiple van genes within a single study centre, which poses a severe challenge to treatment options. The presence of van genes among clinical isolates of S. aureus is a serious concern as the hospital environment acts as a reservoir for the resistance determinants. The emergence of hVISA is a significant threat that requires urgent screening and proper reporting.The present study is retrospective, and this advocates further need for prospective investigation under the umbrella of an antibiotic stewardship programme, thereby adopting control measures to contain this spread and detect resistant strains. This study also signifies that the heterogenic resistance strains upon exposure to vancomycin and teicoplanin at a minimum level could increase MIC significantly, particularly in individuals receiving therapeutic intervention in real time.