Dr.T.V.Rao MD

Professor Of Microbiology

VANCOMYCIN RESISANT

STAPHYLOCOCCUS

Dr.T.V.Rao MD 1

Epidemiology of S aureus Infections

• Predominant reservoir of organisms = human beings

• Approximately 15% – 35% of normal people harbor S aureus in nares or pharynx at a given point. Longitudinal view of carriage:

– 30% prolonged, 50% intermittent, 20% never

– Vaginal carriage in ~10% of premenopausal women

– Rectal and perineal carriage also occur

• Patients with MRSA infections may have high prevalence (60%) of gastrointestinal colonization or carriage

• Organism usually spread by direct person-to-person contact

• Spread from inanimate objects is rare, but has been documented, such as outbreaks among football players, river raft guides, etc.

• Common denominator: repeated trauma in defined area

Sheagren. N Engl J Med. 1984;310:1368-1373.

Rimland et al. J Clin Microbiol. 1986;24:137-138.

Centers for Disease Control (CDC). MMWR Morb Mortal Wkly Rep. 1982;31:605-607. Dr.T.V.Rao MD 2

S. aureus Today • Most common cause of endocarditis (38%)

• Most common cause of nosocomial infection (13%)

• Most common cause of SSI (20%)

• Most common cause of cellulitis, osteomyelitis, septic arthritis

• Common cause of bacteremia, nosocomial pneumonia, foodborne disease, implant infection, abscess, etc

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Methicillin-Resistant S aureus

Historical Aspects 1959 First clinical use of methicillin

1961 First description of MRSA

1967 First report of nosocomial infection in the US (2 cases)

1968 Increase in MRSA in the UK

1968-1979 Rise and subsequent wane of prevalence of MRSA (especially nosocomial infections) in Europe, Australia, and elsewhere (except US)

1975-1980 First reports of problems with MRSA in the US; most occurred in large tertiary care hospitals (especially burn units and ICUs)

1980 MRSA increase in prevalence in US nursing homes; CA-MRSA infections in the US

1998 Emergence of CA-MRSA

Jevons. BMJ. 1961;1:124; Westh et al. Clin Infect Dis. 1992;14:1186-1194; Chambers. Clin Microbiol Rev. 1997;10:781-791; Bradley. Am J Med. 1999;106:2S-10S.

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Staphylococcus and Vancomycin

• Staphylococcus aureus is one of the most common causes of hospital- and community-acquired infections, and treatment of staphylococcal infections is complicated by the ability of this bacterial species to become resistant to antibiotics

• Vancomycin is the drug of choice for therapy of infections due to methicillin (methicillin)-resistant S. aureus (MRSA), but increase in Vancomycin use has led to the emergence of two types of glycopeptide-resistant S. aureus.

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Vancomycin Treatment of MRSA

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Nafcillin Vancomycin

• Time-kill assays Vancomycin kills S. aureus more slowly than beta-lactam antibiotics • Vancomycin treatment of R-sided endocarditis is associated with failure in 15% - 33% vs. 5% for nafcillin • Bacteremia lasts a median of 7-9 days with vancomycin treatment vs. 3-5 days with nafcillin

Small and Chambers, AAC 1990; Korzeniowski et al, Ann Intern Med 1982;Levine et al, Ann Intern Med 1991; Chambers et al Ann Intern Med 1988

Vanc vs B-lactam for MSSA

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S. aureus

Penicillin

[1950s]

Penicillin-resistant

S. aureus

Evolution of Drug Resistance in S. aureus

Methicillin

[1970s]

Methicillin-resistant S. aureus (MRSA)

Vancomycin-resistant

enterococci (VRE)

Vancomycin

[1990s]

[1997]

Vancomycin

intermediate- resistant S. aureus (VISA)

[ 2002 ] Vancomycin

-resistant S.

aureus

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Worldwide Prevalence of MRSA Among

S. aureus Isolates

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Vancomycin Pros and Cons

Disadvantages • Slow bactericidal activity

• Rare VRSA; increasing incidence of VISA and hetero-resistant MRSA

• Low penetration into ELF (10%-30% of serum concentrations)

• Prolonged durations of low serum concentration with current dosing regimens in renally impaired patients

• “Red man” syndrome with rapid infusions; rare neutropenia

Advantages • Low cost

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Beginning of Vancomycin Resistant Staphylocoous

• Vancomycin has been the most reliable therapeutic agent against infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, in 1996 the first MRSA to acquire resistance to vancomycin, was isolated from a Japanese patient. The patient had contracted a post-operative wound infection that was refractory to long-term vancomycin therapy

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Reduced effectiveness of Vancomycin

• A certain group of S aureus, designated hetero-VRSA, frequently generate VRSA upon exposure to vancomycin, and are associated with infections that are potentially refractory to vancomycin therapy. Presence of hetero-VRSA may be an important indicator of the insidious decline of the clinical effectiveness of vancomycin in the hospitals

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Vancomycin - Resistance

• Interesting about vancomycin is that,, resistance to this agent among S. aureus strains took almost 40 years to be recognized, with the first glycopeptide-intermediate S. aureus (GISA) isolate from a pediatric patient in Japan described in 1996 . High-level resistance mediated by the vanA gene complex acquired from vancomycin-resistant enterococci (VRE) emerged in Detroit, Michigan, in 2002 and so far has been limited to the United State

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Mechanism of Vancomycin Resistance

• Vancomycin resistance is acquired by mutation and thickening of cell wall due to accumulation of excess amounts of peptidoglycan. This seems to be a common resistance mechanism for all VRSA strains isolated in the world so far. Dr.T.V.Rao MD 13

Two Component Regulatory Mechanism in Vancomycin Resistance

• A TWO-COMPONENT REGULATORY system VanR–VanS regulates vancomycin resistance in vancomycin-resistant ENTEROCOCCI (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA) strains. VanS is a membrane-associated sensor (of vancomycin) that controls the level of phosphorylation of VanR. VanR is a transcriptional activator of the operon encoding VanH, VanA and VanX. VanH is a dehydrogenase that reduces pyruvate to D-Lac, whereas VanA is a ligase that catalyses the formation of an ester bond between D-Ala and D-Lac.

Dr.T.V.Rao MD 14

Two Component Regulatory Mechanism in Vancomycin Resistance • Vancomycin does not bind to D-Ala-D-Lac, which leads to

vancomycin resistance. VanX is a dipeptidase that hydrolyses the normal peptidoglycan component D-Ala-D-Ala, which prevents it from causing vancomycin sensitivity. VanY is a D,D-carboxypeptidase that hydrolyses the terminal D-Ala residue of late peptidoglycan precursors that are produced if elimination of D-Ala-D-Ala by VanX is not complete. So, D-Ala-D-Lac replaces the normal dipeptide D-Ala-D-Ala in peptidoglycan synthesis resulting in vancomycin resistance VanZ confers resistance to teicoplanin by an unknown mechanism.

• Exploiting genomics, genetics and chemistry to combat antibiotic resistance

• Diarmaid Hughes, Nature Reviews Genetics 4, 432-441 (June 2003) | doi:10.1038/nrg1084

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Regulatory Mechanisms in Vancomycin Resistance

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VISA / GISA •VISA – Vancomycin-intermediate S

aureus (also known as glycopeptide-intermediate S aureus, GISA)

•Resistance = MIC ≥16 μg/mL (Vancomycin-resistant S aureus, VRSA) in the US

• Intermediate resistance = MIC 4 – 8 μg/mL

Sakoulas et al. Clin Infect Dis. 2006;42:S40-S50; Levine. Clin Infect Dis. 2006;42:S5-S12;

Jones. Clin Infect Dis. 2006;42:S13-S24. Dr.T.V.Rao MD 17

Vancomycin Intermediate Resistant Staphylococcus

• The first one, designated Vancomycin intermediate-resistant S. aureus, is associated with a thickened and poorly cross-linked cell wall, resulting in accumulation of acyl-D-alanyl-D-alanine (X-D-Ala-D-Ala) targets in the periphery that sequester glycopeptides

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Vancomycin Resistant Staphylococcus

• The second type, Vancomycin-resistant S. aureus (VRSA), is due to acquisition from Enterococcus spp. of the vanA operon, carried by transposon Tn1546, resulting in high-level resistance

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Change in Defined Breakpoints for Vancomycin

Previous NCCLS Breakpoints for Vancomycin

CLSI Breakpoints for Vancomycin (1/06)

4 g/mL = S 2 g/mL = S

8 – 16 g/mL = I (VISA, GISA) 4 – 8 g/mL = I

32 g/mL = R 16 g/mL = R

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Emerging Glycopeptide Resistance

• High-level resistance to glycopeptides was first reported in enterococci in 1988 , approximately 30 years after the introduction of this antibiotic into clinical practice. Since then, glycopeptide-resistant enterococci have disseminated throughout the world.

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Proportionate Risk with Staphylococcal Resistance

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Factors in glycopeptide Resistance

• To date, seven types of resistance (VanA, -B, -C, D, -E, -G, and -L) in enterococci have been described; these correspond to specific operons (vanA, -B, -C, -D, -E, -G, and -L) responsible for (i) synthesis of a new target (peptidoglycan precursors ending in D-Ala-D-lactate [D-Ala-D-Lac] in VanA, -B, and -D type or D-Ala-D-serine [D-Ala-D-Ser] in VanC, -E, -G, and -L type) having a reduced affinity for glycopeptides and(ii) elimination of the normal D-Ala-D-Ala-terminating precursors

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*Cell-wall thickness is a major contribute to vancomycin resistance

• Mechanism of vancomycin resistance has been extensively studied with the first clinical VRSA strain, Mu50.22–24 Biochemical and transmission electron microscopy (TEM) examination of the Mu50 cell, suggested that it produces increased amounts of peptidoglycan. More murein monomers and more layers (probably 30–40 layers as judged by cell-wall thickness observed with TEM) of peptidoglycan are considered to be present in the cell wall. As a result, more vancomycin molecules are trapped in the peptidoglycan layers before reaching the cytoplasmic membrane where peptidoglycan synthesis occurs

*Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance

Keiichi Hiramatsu The Lancet Infectious diseases

Dr.T.V.Rao MD 24

*Clogging "phenomenon

A higher concentration of vancomycin would be required to saturate all the murein monomers that are supplied at an increased rate in Mu50 (figure 5). Besides the vancomycin-trapping mechanism, designated “affinity trapping,” our recent experiments suggest that the mesh structure of the outer layers of thickened peptidoglycan is destroyed by the trapped vancomycin molecules themselves. This prevents further penetration of vancomycin molecules into the inner part of cell-wall layers (otherwise known as the“clogging”phenomenon) *Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance

Keiichi Hiramatsu The Lancet Infectious diseases Dr.T.V.Rao MD 25

Cell wall thickness Influences the Resistance

• A thickened cell wall, presumably due to the accumulation of increased amounts of peptidoglycan, is the cardinal feature of all the VRSA clinical strains isolated so far from various countries. The cell walls of 16 VRSA strains isolated from seven countries are significantly thicker (mean 31·3 nm, SD 2·6 nm) than the average of vancomycinsusceptible S aureus (VSSA) strains (mean 23·4 nm, SD1·9 nm) as measured by TEM Révertant strains susceptible to vancomycin (MIC<4 mg/L) were obtained from these VRSA strains. They all had decreased cell-wall thicknesses that were indistinguishable from those of VSSA strains

*Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance

Keiichi Hiramatsu The Lancet Infectious diseases

Dr.T.V.Rao MD 26

Investigational Anti-Staphylococcal Antibiotics

• Glycopeptides – Ortivancin (Intermune) – Dalbovancin (Vicuron) – Telavancin (Theravance)

• DHFR inhibitor – Iclaprim (Arpida)

• Novel B-lactams – Ceftobiprole – BMS-247243, RWJ 54428, CB-181963, BAL 5788,

S-3578

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Detection of Vancomycin

Resistance CDC- CLSI based Protocols

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Why Detection of Vancomycin Resistance is Important

• Vancomycin continues to be an important antimicrobial agent for treating infections caused by S. aureus strains that are resistant to oxacillin (MRSA) and other antimicrobial agents. The reduced susceptibility of VISA and VRSA strains to vancomycin leaves clinicians with relatively few therapeutic options for treating these infections.

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Disk diffusion Unreliable in VISA

• VISA isolates are not detected by disk diffusion. Methods that typically detect VISA are non-automated MIC methods including reference broth micro dilution, agar dilution, and Etest® using a 0.5 McFarland standard to prepare inoculum. Automated methods and vancomycin screen agar plates usually detect VISA for which the vancomycin MICs are 8 µg/ml, but further studies are need to define the level of sensitivity of these methods for S. aureus for which the vancomycin MICs are 4 µg/ml.

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Can routine susceptibility tests detect VISA and VRSA?

• Not all susceptibility testing methods detect VISA and VRSA isolates. Three out of six confirmed VRSA isolates were not reliably detected by automated testing systems in a recent report. Subsequently, some manufacturers have optimized their systems for VRSA detection, so laboratories should check with manufacturers to determine if their system has FDA clearance for VRSA detection. VRSA are detected by reference broth micro dilution, agar dilution, Etest®, MicroScan® overnight and Synergies plus™; BD Phoenix™ system, Vitek2™ system, disk diffusion, and the vancomycin screen agar plate [brain heart infusion (BHI) agar containing 6 µg/ml of vancomycin].

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What is the vancomycin agar screen test?

• The vancomycin agar screen test uses commercially prepared agar plates to screen pure cultures of bacteria for vancomycin. These plates contain BHI agar and 6 µg/ml of vancomycin. A 10µl inoculum of a 0.5 McFarland suspension should be spotted on the agar using a micropipette (final concentration=106 CFU/ml). Alternatively, a swab may be dipped in the McFarland suspension, the excess liquid expressed, and used to inoculate the vancomycin agar screen plate

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Quality Control in Detection of Resistance

• For quality control, laboratories should use Enterococcus faecalis ATCC 29212 as the susceptible control and E. faecalis ATCC 51299 as the resistant control. Up to eight isolates can be tested per plate; quality control should be performed each day of testing. Growth of more than one colony is considered a positive result. All staphylococci that grow on these plates should be inspected for purity, and the original clinical isolates should be tested using an FDA-cleared MIC method for confirmation

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Are any modifications required of routine disk diffusion and MIC methods when testing

vancomycin and staphylococci? • Testing should incorporate the following CLSI

recommendations:

• Inoculum: Use direct colony suspension Incubation: 35°C, ambient air, for a full 24 hr. Endpoint: Examine very closely for any indication of growth According to the newest CLSI (formerly NCCLS) standards, a vancomycin-intermediate or –resistant result for staphylococci isolate should be verified by repeating a validated MIC method and the organism identification.

Dr.T.V.Rao MD 34

Infection Control: Conflicting Approaches

• “Search and Destroy”

–Universal application of active surveillance cultures and rigorous enforcement of contact isolation

–Decolonization

• Laissez-Faire

–No cultures, No isolation for pts colonized or infected with MRSA

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Reducing the Vancomycin Resistant Staphylococcus

• It is possible to reduce the selection of vancomycin resistance in MRSA isolates in the hospital by substituting cephalosporins and carbapenems with penicillins that have a relatively strong anti-MRSA activity among beta-lactam antibiotics.

• Therefore, it is necessary to expand antibiotic prescription policy to include beta-lactam antibiotics as well. If we reduce consumption of broad-spectrum cephalosporins (which are ineffective against MRSA), and, this measure combined with effective infection control, the number of MRSA in the hospital would decrease Dr.T.V.Rao MD 36

Emerging option to treat Vancomycin Resistant Staphylococcus

• New agents are being developed against MRSA. Some of them are expected to have considerable activity against hetero-VRSA and VRSA strains as well. Synercid has potent activity against hetero-VRSA and VRSA strains.17 A new quinolone antibiotic, DU-6859a, has MICs of 0·5 and 1 mg/L against Mu3 and Mu50 which are resistant to other quinolones such as levofloxacin, ciprofloxacin, sparfloxacin, and tosufloxacin.47 Linezolid also has good activity against MRSA, and is expected to be useful for cases where vancomycin therapy fails. However, linezolid does not have a cytokilling effect against MRSA. Dr.T.V.Rao MD 37

: Know when to say “no” to

Vancomycin Fact: Vancomycin overuse promotes emergence, selection,

and spread of resistant pathogens.

Actions:

treat infection, not contaminants or colonization

fever in a patient with an intravenous catheter is not a routine indication for vancomycin

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Saving the Precious Drug - Vancomycin

• Decreasing the total consumption of antibiotics both in the hospitals and community, and, using them with good rationale based on accurate diagnosis and susceptibility testing information may help preserve mankind’s precious drug

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Vancomycin resistance – Role of Infection Control Team

• There is significant concern about the spread of VISA and VRSA among patients because of limited treatment options. If a VISA or VRSA is suspected, specific infection control precautions need to be initiated by infection control personnel to decrease the risk of VISA/VRSA transmission to other patients. It is critical for laboratory workers to contact the infection control team immediately when a VISA or VRSA is suspected. Additionally, laboratories should notify the local and/Division of Healthcare Quality Promotion

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Improved Patient Outcomes Associated With Proper Hand Hygiene

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Hand Washing can Still reduce Vancomycin Resistant Staphylococcus spread

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References

1 Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance e Keiichi Hiramatsu The Lancet Infectious diseases

2 CDC Atlanta issues on Staphylocoous resistance to Vancomycin

3 Exploiting genomics, genetics and chemistry to combat antibiotic resistance Diarmaid Hughes, Nature Reviews Genetics 4, 432-441 (June 2003) | doi:10.1038/nrg1084

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Follow me for More Articles of Interest on Infectious Diseases

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Professionals in Developing World • Email

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