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Introduction: Detection of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is currently problematic. Although the population analysis profile with area under the curve (PAP-AUC) is the gold standard for detecting hVISA strains, this method is time consuming. This study aimed to induce vancomycin non-susceptible Staphylococcus aureus isolates in methicillin-resistant S. aureus (MRSA) and to determine the performance of the vancomycin and teicoplanin disk diffusion test for screening of induced and natural vancomycin non-susceptible isolates. Methodology: Vancomycin resistance was induced in vitro in methicillin-resistant S. aureus by serial passage in media with increasing vancomycin concentrations. All test isolates were confirmed for their susceptibility to vancomycin by using a PAP-AUC method. The performance of the vancomycin and teicoplanin disk diffusion test for detecting both induced and natural hVISA/VISA isolates was analyzed using the MedCal program version 10.2.0. Results: The induction test revealed that 42 of 78 MRSA isolates (53.8%) became hVISA and/or VISA. Using 10, 15, 20, 30 µg vancomycin disks and a 30 µg teicoplanin disk, the highest performance (88.9%) for hVISA/VISA detection (71.1%, sensitivity, 100% specificity, 100% positive predictive value, and 75% negative predictive value) was obtained when a 20 µg vancomycin disk was used at 1.0 McFarland inoculum for a 24-hour incubation. Conclusions: The results indicated that using a 20 µg vancomycin disk and bacterial inoculum of 1.0 McFarland is simple to perform and provides a primary result for hVISA/VISA screening within 24 hours.

Background:Staphylococcus aureus, with reduced vancomycin susceptibility, is probably under the regulation of several genes and various express phenotypes.Objectives:This study aimed to investigate the phenotypic differences between vancomycin-susceptible S. aureus (VSSA), vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA) isolates.Materials and Methods:A total of 130 methicillin-resistant S. aureus (MRSA) isolates were studied, including 49 VSSA, 28 hVISA, and 5 VISA isolates from blood cultures and 48 isolates (two VSSA, six hVISA, and 40 VISA) derived in vitro (laboratory-induced/sub-passaged). Their phenotypes were examined using a coagulase tube test, colony spreading on soft agar, and urease activity. The SCCmec and agr typing were performed using multiplex PCR.Results:Most of the MRSA isolates were SCCmec III-agr I (84.5%), followed by SCCmec II-agr II (11.8%). The average plasma coagulation time of vancomycin-non-susceptible isolates was longer than that of the susceptible isolates (12 vs. 2.6 hours). Four hVISA (P = 0.023) and nine VISA (P < 0.001) isolates yielded a negative coagulase test after 24-hour incubation. The percentage of VSSA isolates showing non-spreading colonies (accessory gene regulator (agr) dysfunction) was significantly lower than in the VISA group (P = 0.013), but no significant difference was found between VSSA and hVISA. The VISA group showed higher urease activity than that of the VSSA and hVISA groups (P = 0.002).Conclusions:There were diverse phenotypic changes among vancomycin-non-susceptible S. aureus isolates. This may be due to the variety of related regulatory systems. The diversity of phenotypic expression may result in its misidentification in routine laboratory checks.

Low-level vancomycin-resistant S. aureus designated as heterogeneous vancomycin-intermediate S. aureus (hVISA) have been associated with treatment failure and unable to detect by routine disk diffusion method. As small angle X-ray scattering (SAXS) can be used for studying size, shape, and biology structure of bacterial cells; thus, this study aimed to investigate the SAXS patterns of biomolecules of vancomycin susceptible S. aureus (VSSA), hVISA, and, VISA cells. A total of 9 S. aureus isolates, 3 each from VSSA, hVISA, and VISA groups, were cultured on brain heart infusion agar with and without vancomycin. The cultured cells were kept overnight to reach their exponential phase and subjected to the SAXS using beamline 1.3W: SAXS. Under vancomycin untreated condition, the VISA cells showed different SAXS pattern from those of the VSSA and the hVISA, whereas the vancomycin treated cells of hVISA and VISA displayed similar SAXS patterns. The ribosome of VSSA was significantly smaller than that of hVISA under the untreated condition but showed no statistically different under the treated condition. In addition, when compared the ribosome and the DNA of VSSA with the VISA's and the DNA of VISA with the hVISA's, they were different only under the untreated condition. This preliminary study showed that under the stress condition mediated by vancomycin, the vancomycin non-susceptible S. aureus (hVISA and VISA) had similar SAXS patterns; while under the non-stress condition, the VSSA and hVISA showed similar patterns. This study provides preliminary information on bacterial adaptation under vancomycin-mediated stress condition.