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2. INTRODUCTION:
 Introduced clinically in 1958 for the
treatment of gram-positive bacteria.
 It is developed from Amycolatopris orientalis
[Streptomyces orientalis and later Nocardia
orientalis].
 Complex tricyclic glycopepetide consist of 7
membered peptide chain attached to
disaccharide

4.  Glycopeptides inhibit cell wall synthesis in
dividing bacteria
 The target of glycopeptides is the nascent
pepetidoglycan precursor unit [ lipidII ] emerging
from bacterial cytoplasm
 Vancomycin forms complex with terminal D-
alanine residue of peptidoglycan precursor,
acyl-D-alanyl-D-alanyl moiety ,held firmly via
hydrogen bond

5. Staphylococcus aureus
 One of the most common causes of hospital& community
acquired infections.
 S. aureus was susceptible to pencillin in the early 1940s .
 Increasing resistance to the new semisynthetic penicillinase-
resistant antimicrobial agents (i.e., methicillin, oxacillin, nafcillin,
dicloxacillin). Pencillin resistance developed in 1950s
 Resistance to the penicillinase-resistant penicillins had spread
throughout the world in 1990s .
 This has led to increased reliance on vancomycin for treatment
of methicillin-resistant S. aureus (MRSA) infections.

6.  According to CLSI ,
MIC of hVISA is 1-4m𝑔/𝑙
VISA is 4-8𝑚𝑔/𝑙
VRSA is ≥ 16𝑚𝑔/𝑙
 Staphylococcus with susceptible range MICs have another form of
decreased susceptibility to vancomycin , called heteroresistant or
hVISA
 These are the strains with varying levels of resistance to vancomycin
 hVISA are precursors of VISA
 They are similar but with pronounced changes and associate with
mutation control genes

7. hVISA/VISA
 First hVISA strain [mu3] &First VISA
strain[mu50] reported in japan in 1996
 More than 21 VISA strain isolated
 VISA strains have lower growth rate and
thicker cell wall
 hVISA produce 3-5 fold grater quantities of
PBP2 and 3-8 fold increased quantities of
cell wall precursor than VISA

8. MECHANISM OF RESISTANCE:
It is a subject of active investigation
 Common feature of VISA is thickened cell wall due to enhanced
availability of D-alanyl-D-alanine .
 Thickened cell wall may trap vancomycin within peptidoglycan
and utilized for trans peptidation and trans glycosylation
They have
 increased cell wall turn over,
 decreased autolytic activity,
 decreased agr functionality

9.  Vancomycin resistance developed in VISA is
due to loss of mutational control genes.
 The agr operon induces the production of
virulent factors
 Under vancomycin exposure, agr function loss
provides selection of hVISA/VISA strains
 As a result of loss of agr function
 Increase bio film production
 Decrease autolysis
 Thicker cell wall

10.  Population analysis profiling[PAP]:gold std
method
 Inoculate test isolate and Mu3 strain
(S.aureus ATCC 700698) in trypticase
soya broth (TSB)
 incubate at 37°C for 24 hours.

11.  Dilute the culture in saline to 10-3& 10-6
and inoculate on to brain heart infusion
agar (BHIA) plates containing 0.5, 1, 2,
2.5, 4 mg/L vancomycin and incubate at
37°C for 48 hours.
 After 48hrs of incubation ,The no of colonies
counted and plotted against the vancomycin
concentration on a semilogarithmic graph

12.  The graph obtained was used to calculate
area under curve[AUC] for each isolate
 Ratio between AUC of test isolate to the
AUC of reference strain MU3 was calculated
 The isolate was identified as hVISA/VISA if
ratio of AUC was 0.9 − 1.3 and ≥
1.3 respectively

14.  Simplified population analysis
10μl of 108cfu/ml into BHI with 4μg vancomycin
 Growth at 24 hr- potential VISA
 Growth at 48 hr-potential hVISA
 Conformed VISA- having MIC 8μg/ml
 Conformed hVISA-stable for >9 days in drug free
medium
 VISA isolates are also resistance to teicoplanin
 Inoculating MHA plate with 5𝜇𝑔 𝑡𝑒𝑖𝑐𝑜𝑝𝑙𝑎𝑛𝑖𝑛 is a
screening method

15.  E -Test GRD:
 Double sided vancomycin and teicoplanin
strip placed on MHA with 5% sheep blood
agar.
 Incubated at 35℃ for 24 and 48 hr
 If MIC is ≥ 8𝜇𝑔/𝑚𝑙 GRD test positive for
hVISA

16.  BHI Screen agar plates:
 BHI agar with 16g/l pancreatic digest of casein and
4μ𝑔/𝑚𝑙 𝑣𝑎𝑛𝑐𝑜𝑚𝑦𝑐𝑖𝑛
 Two inocula prepared:0.5McFarland and
2.0McFarland.
10𝜇𝑙 𝑓𝑟𝑜𝑚 𝑒𝑎𝑐ℎ 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑎𝑟𝑒 𝑑𝑟𝑜𝑝𝑝𝑒𝑑 𝑖𝑛𝑡𝑜 𝑡ℎ𝑒 𝑝𝑙𝑎𝑡𝑒
 Allowed to dry for 5 min and incubated at 35℃ and
examined at 24 hr and 48hr
 Individual colonies in each drop are counted.

17.  VISA can be detected by automated MIC
methods [VITEK, microscan(rapid
&conventional)]
 VISA isolates are not detected by disk diffusion
because zone diameters produced by VSSA
and VISA strains are indistinguishable.
 Vancomycin screen agar plates usually detect
VISA .
 BHI agar supplemented with vancomycin
hydrochloride at a concentration of 6μg/ml.

18.  Prepare a suspension equivalent to 0.5
McFarland standard
 Spot inoculate
 Incubate at 35ºC for 24hours
 Read the plates in transmitted light
 TREATMENT:
 Drugs such as linezolid,
daptomycin,Telavancin,Ceftaroline

19. VRSA
 Decrease susceptibility to vancomycin
define true ‘vancomycin resistance’ [MIC-
≥ 16]
 The strain are aquired from enterococcus
VanA gene
 VanA gene transferred from enterococcus
to MRSA isolates result in replication of
actual enterococcal VanA plasmid in new
staphylococcus host
 Tn1546 could transpose from the
incoming plasmid to a resident plasmid
in the recipient.
 The acquired plasmid behaves as a
suicide gene delivery vector.

21.  Van A gene produce dipeptide D-ala-D-lac in
the place of D-ala-D ala to which drug
cannot bind.
 Nine VRSA strains isolated in the United
States
 Three (VRSA-3, 5, and 6) maintained the
enterococcal vanA plasmid
 whereas in VRSA-1, 7, 8, 9, and 10,Tn1546
moved from the enterococcal plasmid to a
staphylococcal plasmid.

23.  Two systems Van S and VanR
 Van S:Controls phosphorylation of Van R
 Van R: Activates Van H ,VanA,VanX
Van H: Reduce pyruvate to D- lac
VanA: Catalyse formation of ester bond
between D-ala and D-lac
Van X: Hydrolyze D-ala-D-ala and prevent
vancomycin sensitivity
VanY: D,D carboxypeptidase that hydrolyse
terminal D-ala residue

24.  VanZ: Resistance to tecioplanin
 CLOGGING PHENOMENON:
Prevention of penetration of trapped
vancomycin in the inner part of cell.

25.  Automated methods [microscan, vitek, BD
Phoenix]
 Broth microdilution,
 Agar dilution,
 Gradient diffusion,
 Vancomycin screen agar plates [brain heart
infusion (BHI) agar containing 6 μg/ml of
vancomycin ]
 Two new method: Nitrate reductase assay and
Rezasurin microplate method

26.  RMM: Performed on 96 wells .
 100μl suspension is added to wells containing drug
 Incubated at 35℃ After 5 hr of incubation add
rezasurin[30𝜇𝑙 𝑜𝑓0.02%] 𝑎𝑛𝑑 𝑟𝑒𝑖𝑛𝑐𝑢𝑏𝑎𝑡𝑒𝑑 𝑓𝑜𝑟 1ℎ𝑟.
 MIC is the lowest concentration of drug prevent the
change in colour

27.  NRA: Cation adjusted Muller Hinton broth
with 1000𝜇𝑔/𝑚𝑙 potassium nitrate is used.
 Same as that of RMM method .after 5 hr of
incubation, add 50 𝜇𝑙 griess reagent
 After 5 min red/purple colour developed
 MIC is the lowest concentration of drug
without colour change

28.  RISK FACTORS
 Exposure to vancomycin(or other
glycopeptide antibiotics) stands out as a
strong risk
 The isolates are rare
 Renal failure .

29.  Microbiology of VISA and VRSA
 Morphologically, colonies of VISA and VRSA
isolates often look smaller than their susceptible
counterparts
 Vancomycin resistant strains may require more
incubation time for coagulase detection. If the
coagulase reactions are incubated for less than 4 h,
the result may be falsely negative and the isolate
maybe misclassified as coagulase-negative
staphylococci.
 Thus, for any staphylococcus with suspected
vancomycin resistance,the coagulase test should
be incubated for more than 4 h

30. CLINICAL MICROBIOLOGY REVIEWS, July 2002, p. 430–
438 Vol. 15, No. 3 Arjun Srinivasan,1* James D. Dick,2 and
Trish M. Perl1
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov.
2009, p. 4580–4587
Mandell,douglas and bennetts Principle&practice of
infectious disease.
Journal from american society of microbiology 2003,october