VANCOMYCIN RESISTANT ENTEROCOCCI

1. VANCOMYCIN RESISTANT ENTEROCOCCI Dr.T.V.Rao. MD Dr.T.V.Rao MD

2. Introduction to Enterococci • Enterococci are gram-positive cocci which often occur in pairs (diplococci) • Two species are common commensal organisms in the intestines of humans: E. faecalis and E. faecium • Enterococci occur almost everywhere, including soil, food, water, plants, animals, birds, and insects they inhabit in humans and other animal's gastrointestinal tract and the female genital tract Dr.T.V.Rao MD

3. Characters of Enterococci • Gram(+) , Catalase(－) Cocci • Can grow in media : 6.5% sodium chloride • E. faecalis and E. faecium (90%) • Part of the normal bowel flora. the prominent cause of nosocomial infections. Dr.T.V.Rao MD

4. Habitat of Enterococci • Enterococci normally inhabit the bowel. They are found in the intestine of nearly all animals, from cockroaches to humans. Enterococci are readily recovered outdoors from vegetation and surface water, probably because of contamination by animal excrement or untreated sewage . In humans, typical concentrations of enterococci in stool are up to 108 CFU per gram . Dr.T.V.Rao MD

5. Growing Importance of Enterococci • Enterococcus species are normal flora of the intestinal tract. Enterococcus faecalis frequently causes infections within the peritoneal cavity, especially following penetrating trauma such as gunshot wounds, and surgical wounds, urinary tract infections, prostate infections, and infections of damaged or compromised skin, such as diabetic or decubitus ulcers, burns, and surgical wounds. Other opportunistic fecal streptococci include E. faecium and E. durans. Dr.T.V.Rao MD

6. Prominent Cause of Nosocomial Infections • The enterococci have become the second most common bacterium isolated from nosocomial urinary and wound infections, and the third most common cause of nosocomial bacteremia. Furthermore, the enterococci are among the most antibiotic resistant of all bacteria, with some isolates resistant to all known antibiotics Dr.T.V.Rao MD

7. Clinical Manifestation • Infections with VRE do not differ from other enterococcal infections other than in their therapy. • The most common sites of infection : The urinary tract and bloodstream. • In addition, enterococci may cause endocarditis due to their ability to adhere to heart valves. • They rarely cause respiratory tract infections. Dr.T.V.Rao MD

8. Glycopeptides Mechanism of Action • Vancomycin and teicoplanin inhibit cell wall synthesis by forming complexes with peptidyl-D-alanyl-D-alanine termini • vanA and vanB resistance phenotypes are associated with the acquisition of gene clusters that lead to the production of peptidoglycan ending in D-alanyl-D-lactate Dr.T.V.Rao MD

9. Vancomycin Resistance Increases Morbidity and Mortality • Vancomycin-resistant enterococci (VRE), first reported in Europe in 1988, are emerging as a global threat to public health . The incidence of VRE infection and colonization among hospitalized patients has increased rapidly in the last 7 years. From 1989, the year VRE was first identified in the United States, through 1993. Infection with VRE may be associated with increased mortality , and no effective antimicrobial therapy is available for many VRE . Dr.T.V.Rao MD

10. VRE Epidemiology • Found world-wide, but rates vary greatly • Hospital outbreaks often involve clonal spread • Also seen in nursing homes and long term care facilities • In Europe, animals may be a source due to the use of the glycopeptide, avoparcin • First described in Europe • Primarily a nosocomial pathogen • Alarming increase from 1989 to 1993 • intensive care units teaching hospitals hospitals with more than 500 beds. Dr.T.V.Rao MD

11. Vancomycin and Development of VRE Strains • Mechanism of action: Inhibits bacterial cell wall synthesis • Spectrum of action: Gram positive organisms • Including: Listeria, Rhodococcus, Peptostreptococcus • Bacteriostatic against enterococcus • Mechanism of resistance: • Enterococcus: Van A – E • Peptidoglycan precursor has decreased affinity for vancomycin – D-ala-D-ala replaced by D-ala-D-lac Dr.T.V.Rao MD

12. Vancomycin-Resistant Enterococci • (VRE), are bacterial strains of the genus Enterococcus that are resistant to the antibiotic vancomycin. Enterococci are gram-positive coccoid-shaped bacteria found in the digestive tract of some humans. To become VRE, vancomycin-sensitive enterococci typically obtain new DNA in the form of plasmids or transposons which encode genes that confer vancomycin resistance. Dr.T.V.Rao MD

13. vanA and vanB Phenotypes

14. MOLECULAR BASIS OF VANCOMYCIN RESISTNACE

15. vanC, vanD, and vanE Phenotypes

16. Glycopeptide-Resistance Transposons • In E. faecium, vanA and related genes are located on a transposon (Tn1546) which resides on a plasmid • Resistance is associated with a number of genes (vanHAX gene cluster, vanS, vanR, vanX and vanZ) • vanB phenotype is associated with Tn5382 Dr.T.V.Rao MD

17. vanA Genes and Their Enzymatic Products (I) • vanA protein is a ligase • produces D-ala-D-lac rather than D-ala-D-ala (vanA alone does not lead to resistance) • vanH protein is a dehydrogenase • converts pyruvate to D-lactic acid (the source of lactate for the above reaction) • vanX protein is D,D-dipeptidase • cleaves D-ala-D-ala (Cetinkaya et al. 2000. Clin Micro Rev. 13: 686-707) Dr.T.V.Rao MD

18. vanA Genes and Their Enzymatic Products (II) • vanS is a sensor • detects the presence of vancomycin or some effect of it • vanR is a regulator • may turn on vanHAX • vanY is a carboxypeptidase • cleaves terminal D-Ala • vanZ increases the MIC of teicoplanin • mechanism is unknown Cetinkaya et al. 2000. Clin Micro Rev. 13: 686-707 Dr.T.V.Rao MD

19. vanB Genes and Resistance • vanB is also a ligase • involved with D-ala-D-lac production • vanXB has dipeptidase activity • associated with high level vancomycin resistance • vanHB, vanYbB, vanSB and vanRB genes • similar to their vanA counterparts • Teicoplanin does not induce the synthesis of vanB proteins and cells are still susceptible Cetinkaya et al. 2000. Clin Micro Rev. 13: 686-707 Dr.T.V.Rao MD

20. vanC, vanD and vanE Resistance • vanC ligases result in D-ala-D-ser • net result is reduced vancomycin binding • vanD ligase • has some homology with other van ligases • vanE ligase • rare • more closely related to vanC than the other van ligases Dr.T.V.Rao MD

21. ResultSpreading Resistance • Enterococci that acquire the vanA phenotype are highly resistant to vancomycin and to teicoplanin • Enterococci can pass the vanA gene cluster to S. aureus • E. faecalis rather tan E. faecium (so far) Dr.T.V.Rao MD

22. Infections Caused by Vancomycin Resistant Enterococci • Urinary tract infection (most common) • Intra-abdominal and pelvic infection (also common) • Surgical wound infection • Bacteremia—bacteria in the blood • Endocarditis —infection of the inner surface of the heart muscles and valves • Neonatal sepsis —bacteria in the blood, occurring in infants • Meningitis —infection of the membranes that surround the brain and spinal cord Dr.T.V.Rao MD

23. DIAGNOSIS OF DRUG RESISTANCE INENTEROCOCCI Dr.T.V.Rao MD

24. Diagnosis of VRE Specimens can proceeded as per clinical Needs • Diagnosis requires culturing the organism. VRE is easily grown on culture plates in a laboratory. To get material to culture, a sample of the infected tissue is taken. For a wound infection, a swab is usually rubbed over the surface to get infected material. Blood is drawn and cultured to detect sepsis or endocarditis. Urine samples are taken to identify urinary infections . Dr.T.V.Rao MD

25. Enterococci are Bile Esculin Test Positive • Enterococci are able to grow in the presence of bile and hydrolyze the esculin; the liberated diphydroxycourmarin complexes with ferric citrate present in the media to form a dark brown/black soluble compound. Dr.T.V.Rao MD

26. Bile Esculin Test Positive for group D streptococci and enterococci Dr.T.V.Rao MD

27. Detection of Vancomycin Resistance • Susceptibility to vancomycin was performed by Kirby-Bauer Disc Diffusion Method on Mueller Hinton Agar by using 30µg vancomycin disc . Vancomycin resistance was also determined by Vancomycin agar screen method using 6µg/ml of vancomycin incorporated in Brain Heart Infusion (BHI) agar. Dr.T.V.Rao MD

28. Detection of Vancomycin Resistance • Minimum Inhibitory Concentration (MIC) of all the isolates were done by Macro broth dilution method, using dilutions of vancomycin ranging from 2 µg/ml to 512 µg/ml. Dr.T.V.Rao MD

29. Drug Resistance can be Established by E-Test Dr.T.V.Rao MD

30. *Chromogenic Methods in Diagnosis of VRE • Chromogenic medium for the detection of Vancomycin Resistant Enterococcus (VRE) E. faecalis and E. faecium • * Colorex™ Prepared Chromogenic Media by BioMed Diagnostics Dr.T.V.Rao MD

31. Genotypic Detection of VRE • Rapid detection of vancomycin resistance by polymerase chain reaction (PCR). useful in epidemiologic studies • PCR cant be performed directly on clinical specimens. Dr.T.V.Rao MD

32. Control and Prevention • Limiting the use of certain broad spectrum antibiotics may also lead to a decrease in the rates of VRE colonization and infection. • One study suggested that reduction of third-generation cephalosporins with the substitution of piperacillin/tazobactam could reduce the incidence of VRE in an intensive care unit setting Hospital Infection Control Practices Advisory Committee (HICPAC). Recommendations for preventing the spread of vancomycin resistance. Infect Control Hosp Epidemiol 1995; 16:105 Dr.T.V.Rao MD

33. Control and Prevention • The CDC has recently published recommendations for preventing the spread of vancomycin resistance • Prudent use of vancomycin • Education of hospital staff regarding the problem • Rapid and accurate identification of VRE in the microbiology laboratory • Aggressive infection control measures utilizing contact isolation and cohorting where necessary to prevent person-to-person transmission Hospital Infection Control Practices Advisory Committee (HICPAC). Recommendations for preventing the spread of vancomycin resistance. Infect Control Hosp Epidemiol 1995; 16:105 Dr.T.V.Rao MD

34. Hand Washing can Reduce the Spread of VRE Dr.T.V.Rao MD

35. Created by Dr.T.V.Rao MD for ‘ e ‘ Learning Resources for Medical and Paramedical Professionals in Developing World • Email • doctortvrao@gmail.com Dr.T.V.Rao MD