Methicillin-resistant Staphylococcus aureus with
Reduced Susceptibility to Vancomycin in
Sanprasitthiprasong Hospital
Pawana Panomket PhD*, Suthinee Thirat MD*
Surasak Wanram PhD*, Rungnapa Pankla Sranujit PhD**
* College of Medicine and Public Health, Ubon Ratchathani University, Ubon Ratchathani, Thailand
** Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand
Background: Staphylococcus aureus is a species of bacteria that causes a number of diseases and more than 60% of it is
presently resistant to methicillin. Vancomycin is the drug of choice for the eradication of methicillin-resistant S. aureus
(MRSA).
Objective: This study aimed to investigate the susceptibility of heterogeneous vancomycin intermediate S. aureus (hVISA) and
vancomycin intermediate S. aureus (VISA) to vancomycin by standard disk diffusion, microbroth dilution, a one-point
population assay, and a population analysis profile.
Material and Method: Sixty-eight MRSA isolates from patients admitted to Sanprasitthiprasong Hospital between November
2010 and November 2011 were tested.
Results: Standard disk diffusion showed that all the MRSA isolates were susceptible to vancomycin. Vancomycin MICs for all
isolates were 1-2 μg/mL. Only two MRSA isolates (2.9%) were able to grow on brain heart infusion agar supplemented with
vancomycin 4 μg/mL and were confirmed by a population analysis as hVISA.
Conclusion: This study showed the effect of vancomycin on MRSA and the need for early detection and controlled planning.
Keywords: Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin, Heterogeneous vancomycin-intermediate S.
aureus (hVISA), Vancomycin-intermediate S. aureus (VISA), High level vancomycin-resistant S. aureus (VRSA)
Staphylococcus aureus is a gram-positive
coccus that can cause infections in several systems,
such as the respiratory and urinary tracts, and in
the blood and open wounds. Most S. aureus are
methicillin-resistant (MRSA) and the preferred drugs
for the treatment of these are glycopeptides, such as
vancomycin and teicoplanin.
Several research studies found MRSA with
reduced susceptibility to vancomycin, the first being
the work in Japan by Hiramatsu et al. The isolates in
this study were vancomycin-intermediate S. aureus
(VISA)(1). Currently, the mechanism of intermediate
resistance to vancomycin in S. aureus is unknown.
The transfer of the vanA gene-resistant determinants
from vancomycin-resistant enterococci (VRE) to S.
aureus by cell-to-cell mating was demonstrated in
vitro(2). However, none of the VISA strains was shown
to have any van determinants that were present in
VRE(3). VISA strains had lower growth rates and thicker
cell walls(4) and produced three to five times more
penicillin-binding proteins 2 and 2’ and three to eight
times more cell wall precursors in comparison to
susceptible strains(5).
There have been reports of the prevalence of
heterogeneous vancomycin-intermediate S. aureus
(hVISA) ranging from 0-74%(3,6). hVISA, namely Mu3,
was first reported in 1997 in Japan(7) and classified by
the Criteria of Clinical Laboratory Standards Institute
(CLSI) as having a vancomycin broth microdilution
MIC in the susceptible range. The isolate contained
sub-populations that were able to grow in media
containing 5 to 9 μg/mL of vancomycin. VISA or Mu50
was recovered several months later in the same hospital
in Japan(7) and both strains were suggested to be closely
related. Hiramatsu et al initially discovered hVISA, then
VISA, and finally high level vancomycin resistant S.
aureus (VRSA)(8). In Thailand, S. aureus with reduced
susceptibility towards vancomycin was first reported
S8 J Med Assoc Thai Vol. 97 Suppl. 4 2014
by Trakulsomboon et al(9). They found three isolates
of VISA by a one-point population analysis and later
reported VRSA. In addition, a study in Srinagarind
Hospital found 7 hVISA by a population analysis profile
curve(10).
The present study that is the subject of this
paper took place in Sanprasitthiprasong Hospital, a
medical school with a high rate of MRSA infection and
a high use of vancomycin therapy.
Material and Method
Bacterial strains
S. aureus isolates were obtained from
patients admitted to Sanprasitthiprasong Hostpital from
November 2010 to November 2011. All isolates were
tested by 1 μg oxacillin disk, oxacillin resistance
screening agar (Oxoid, Basingstoke, Hants, UK), and
oxacillin microbroth dilution (Trek diagnostic systems,
Biosciences Inc., Magellan, USA). S. aureus isolates
that were resistant to oxacillin by all methods were
selected for further study. Sixty-eight MRSA isolates
from blood (36 isolates), respiratory secretions and
sputum (22 isolates), and pus (10 isolates) were included
in the present study.
Standard disk diffusion for vancomycin
The disk diffusion test with 30 μg vancomycin
was performed on Mueller Hinton agar (Hardy
diagnostics, Santa Maria, USA) using the Kirby-Bauer
method. S. aureus ATCC 25923 was used as a
vancomycin-sensitivity control with an expected
inhibition zone of between 18 and 23 mm. The results
were interpreted according to CLSI(11). The inhibition
zone of a susceptible (S) organism was considered to
be >15 mm.
Minimal inhibitory concentration (MIC) for
vancomycin
MIC was tested by Sensititre gram-positive
plate (Trek diagnostic systems, Biosciences Inc.,
Magellan, USA) and performed according to the
manufacturer’s instructions. The concentrations of
vancomycin were 0.25-32 μg/mL. Isolates with
vancomycin MIC of <2 μg/mL were interpreted as
susceptible as recommended by CLSI(11).
One-point population assay
A one-point population assay was performed
by the method of screening agar(7,12). Briefly, 100 μL of
bacterial suspension adjusted to McFarland No. 0.5
was spread onto brain heart infusion (BHI) agar (Hardy
diagnostics) supplemented with 4 μg/ml of vancomycin
(Merck KGaA, Darmstadt, Germany). Plates were
incubated at 35°C for 24-48 hours. S. aureus strains
Mu3 (hVISA) and Mu50 (VISA) were used as positive
controls. S. aureus ATCC 29213 was used as the
negative control strain.
Population analysis profile (7,13)
The bacterial suspension was adjusted to an
optical density of 108 CFU/ml and serial 10-fold dilutions
were spread over brain heart infusion (BHI) agar plates
containing vancomycin at concentrations ranging from
1 to 64 μg/mL. After inoculation at 35°C for 48 hours,
the number of viable colonies was counted. S. aureus
strains Mu3, Mu50, and ATCC 29213 were used as
controls. The number of resistant cells contained in 1
ml of the starting cells suspension was calculated and
plotted on a semi-logarithmic scale.
Results
Standard disk diffusion and MIC
Through the use of standard disk diffusion, it
was found that all isolates were susceptible to
vancomycin with inhibition zones ranging from 16 mm
to 24 mm. Vancomycin MIC for all isolates ranged
from 1 μg/mL to 2 μg/mL, which were interpreted as
susceptible(11).
One-point population assay
Of the 68 MRSA isolates, only two isolates
(M005 and M102) grew on BHI supplemented with 4
μg/mL of vancomycin after 24-h incubation.
Population analysis profile
Isolates M005 and M102 had hVISA curves
by a population analysis profile versus the reference
strains (Fig. 1). Calculation of the areas under the curves
(AUC) showed Mu3 was 19.7, M005 was 19.05, and
M102 was 19.65. The ratio of the AUC of the test were
0.97 and 0.99, respectively. Mu50 was 51.90 and gave
an AUC ration of 2.63. The vancomycin MIC for both
hVISA isolates were 2 μg/mL.
Discussion
In the present study, two of the 68 MRSA
isolates were hVISA that were susceptible to
vancomycin by both standard disk diffusion and
microbroth dilution. They were isolated from sputum
of different hospitalized patients. These patients were
admitted for pneumonia, had never been treated with
vancomycin before, and were treated with ceftriaxone.
J Med Assoc Thai Vol. 97 Suppl. 4 2014 S9
M005 was resistant to tetracycline, erythromycin, and
ciprofloxacin by standard disk diffusion and microbroth
dilution. By the time, M102 was resistant to
erythromycin and ciprofloxacin but susceptible to
tetracycline. However, both M005 and M102 were
susceptible to tigecycline.
This was Sanprasitthiprasong Hospital’s first
warning about MRSA, an important hospital pathogen
with reduced susceptibility to vancomycin. The concern
over the extent of hVISA was that it was not detectable
with standard disk diffusion and microbroth dilution.
Therefore, these isolates were not known to have
reduced susceptibility to vancomycin in clinical
microbiology laboratories. Moreover, vancomycin
treatment clinically failed regarding hVISA strains(4,7,14).
The Mu3 strain was serially passaged in
increasing concentrations of vancomycin in vitro,
giving rise to sub-populations with levels of resistance
comparable to that of Mu50. This phenomenon
suggests that colonization or infection with VISA or
hVISA, with repeated vancomycin exposure, acts as a
selection pressure on the development of the resistant
population(15). Thus, incorrect identification of hVISA
or VISA may increase the incidence of resistant strains.
This poses a problem for future anti-microbial therapy
and patients infected with these organisms may fail to
respond to treatment.
The identification of hVISA is important in a
clinical microbiology laboratory. Multiple studies
demonstrated high sensitivity and specificity in hVISA
detection. However, variations between laboratories
were observed by the Macro E-test(12,16-18). A population
analysis profile sets a high standard for hVISA
detection but is labor intensive. In this study, hVISA
was detected by a one- point population assay. The
susceptible strain was not able to grow in media
containing 4 μg/mL vancomycin. However,
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