MRSA (Methicillin resistant staphylococcus aureus)

resistance against methicillin

1. Methicillin Resistant Staphylococcus aureus (MRSA) Presented by Nagaraj.s M.Sc. 3 rd year microbiology.

2. Carbapenems penicillins cephalosporins monobactams enzymes altered binding sites efflux pumps (beta-lactamase) (PBP PBP2a) Erwinia , Neisseria, E.coli mecA , mecC genes of staphylococcus Beta-lactam ring

3. Staphylococcus

4. Taxonomy Domain : Bacteria Phylum : Firmicutes Class : Bacilli Order : Bacillales Family : Staphylococcaceae Genus : Staphylococcus

5. Introduction Staphylococci are gram positive cocci that occur in grape like clusters. They are ubiquitous and most common cause of localised suppurative lesions in humans. Their ability to develop resistance to penicillin and other antibiotics enhance their importance as a human pathogen, especially in the hospital environment. Staphylococci was first observed in human pyogenic lesions by von Recklinghausen in 1871. Sir Alexander Ogston who gave it name Staphylococcus ( staphyle = bunch of grapes, kokkos = berry). He noticed that Non-virulent Staphylococci were also often present on skin surfaces.

6. Cultural characteristics They grow readily on ordinary media with in a temperature range of 10 to 42c. The optimum being 37c and a pH of 7.4-7.6. They are aerobes and facultative anaerobes. On Nutrient agar , it produces circular, opaque, smooth, shiny, and easily emulsifible. Most strains produce golden yellow pigment. Pigment production is enhanced by 1% glycerol monoacetate or milk is incorporated in the medium. On Nutrient agar slope , confluent growth presents a characteristic oil-paint appearance. On Blood agar , most strains are haemolytic(beta-haemolytic), especially when incubated under 20-25% of CO2. In Mac Conkey agar , they produce lactose fermenting pink coloured colonies. In liquid media , uniform turbidity is produced. Selective media are Ludlam’s media (LiCl 2 and tellurite), Salt milk agar , salt broth .

7. Antigenic structures Cell associated polymers Polysaccharide peptidoglycan layer Teichoic acid Capsular polysaccharide Cell surface proteins Protein A Clumping factor

8. Antigenic structures Extracellular enzymes Coagulase Lipid hydrolases Hyaluronidase Heat stable nuclease Protein receptors

9. Toxins Cytolytic toxins Alpha haemolysin Beta haemolysin Gamma haemolysin Leucocidin (PVL) toxin Enterotoxin Toxic shock syndrome Exfoliative toxin

10. Confirmatory tests for S.aureus Deoxyribose nuclease test Thermostable endonuclease test Mannitol fermentation test AccuProbe staphylococcus aureus test (Gen-probe)

11. MRSA (METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS )

12. Emergence of methicillin-resistant S. aureus (MRSA) Penicillin was the first beta-lactam antibiotic developed for the treatment of S. aureus infections. Which is usually fatal . 1 The introduction of penicillin to treat infections caused by S. aureus greatly improved the prognosis of patients with severe staphylococcal infections. 12 these strains resistant to penicillin soon appeared following its clinical use . 12 Penicillin resistance is due to the production, by the bacteria, of penicillinase, which inactivates the antibiotic. Penicillinase hydrolyses the beta-lactam ring that is central to the antimicrobial activity of these drugs. The emergence of penicillin resistance in S. aureus stimulated the development of new antibiotics such as streptomycin, tetracycline, erythromycin, and chloramphenicol in the 1950s. 12

13. These new antibiotics were developed and put into clinical use, resistance to them also appeared. 4 The continuous search for antibiotics active against penicillin-resistant S. aureus led to the development of methicillin also known as methicillin or Staphcillin . 5 Methicillin is a semisynthetic derivative of penicillin, developed in the late 1950s, by a modification of the penicillin structure which conferred resistance to penicillinase. 9 it kills bacteria by inhibiting bacterial cell-wall synthesis, a mechanism of action similar to that of other penicillins . Methicillin resistance occurs due to the acquisition of mecA or mecC gene by previously susceptible strains. 6 The mecA gene codes for an altered penicillin-binding protein called penicillin-binding protein-2a (PBP2a) with reduced affinity to the entire beta-lactam class of antibiotics including penicillin, cephalosporin and carbapenems 11 except the recently approved beta-lactams, ceftaroline and ceftobiprole. 7

14. mecC carrying MRSA was identified in 2007, a retrospective search of archived collections found mecC in isolates collected as early as 1975. indicating that these strains have been around for a long time possibly as long as the mecA -MRSA strains. 6 MecC gene is a homolog of mecA . It was initially designated mecALGA251. It shares 69% nucleotide homology with mecA. 6 MRSA isolates carrying mecC have been isolated from human as well as animal hosts. 8 the mecA PCR or PBP2a latex agglutination test fails to detect mecC . In sensitivity testing, using both cefoxitin and oxacillin, mecA -MRSA show resistance to both antibiotics whereas the majority of mecC MRSA will express resistance only to cefoxitin. 8 This discrepancy is explained by the observation that PBP2a produced by mecC strains have higher affinity to oxacillin than cefoxitin. 8

15. Types of MRSA

16. Types of MRSA When MRSA strains first occurred, they were usually confined to elderly patients admitted to healthcare facilities especially those with previous antibiotic use. The MRSA strains were also isolated from apparently healthy individuals in the communities of no previous contact with healthcare facilities. MRSA strains circulating in the healthcare settings and the community were classified as either nosocomial or healthcare associated MRSA (HA-MRSA) and community- associated MRSA (CA-MRSA). 10 This was followed by a new type of MRSA that arose from animals, designated Livestock-associated MRSA (LA-MRSA) has recently been described. 10

17. Healthcare-associated MRSA Healthcare-associated MRSA (HA-MRSA) were isolated from patients admitted to healthcare facilities such as nursing homes and long-term care facilities. the infections caused by HA-MRSA include bloodstream infections, urinary tract infections, respiratory tract infections, surgical-wounds and device-associated infections. 2 Risk factors for acquiring HA-MRSA include previous admission to healthcare facilities, impaired immune system, use of multiple antibiotics, use of invasive medical devices and old age. 11 Genetically, the HA-MRSA carried SCC mec types I, II and III, are usually multi resistant to antibiotics, and tend to multiply slowly in culture. 11

18. Community-associated MRSA (CA-MRSA) Community-associated MRSA (CA-MRA) strains were initially reported in the late 1980s among individuals living in remote communities in Western Australia with no previous history of hospitalization. 9 CA-MRSA were mostly associated with skin and soft tissue infections such as impetigo, cellulitis, folliculitis and boils and those at risk were the young patients. 10 some CA-MRSA strains have been reported to cause severe infections including necrotizing fasciitis, post-influenza pneumonia, septic thrombophlebitis, septic arthritis, and bacteremia . 10 CA-MRSA are usually susceptible to non-beta lactam antibiotics carry smaller-sized SCC mec types IV, V and VI. 10 CA-MRSA strains often express lower levels of resistance to oxacillin (MIC; 8–32 mg/L) and multiply faster than HA-MRSA strains with significantly shorter doubling times which may help CA-MRSA achieve successful colonization by enabling it to out compete commensal bacterial flora. 10

19. Livestock-associated MRSA Staphylococcus aureus is also an important cause of infections in live stock resulting in economic losses in the food industry. 11 Livestock-associated MRSA (LA-MRSA) strains were initially identified because they were non-typeable by pulsed-field gel electrophoresis following digestion with SmaI restriction enzyme. 12 The molecular typing revealed that LA-MRSA defined to a new lineage of MRSA that belonged to clonal complex 398 (CC398). 16 the LA-MRSA ST398 was initially reported among livestock, 11,12 it has also appeared in the community among human patients in contact with infected or colonized animals which is considered the major risk factor for LA-MRSA colonization. 12 Other LA-MRSA lineages reported in humans include ST9, ST97 and ST433. 18 LA-MRSA has also caused invasive infections including endocarditis, osteomyelitis, and ventilator-associated pneumonia in humans. 13,14

20. By applying epidemiological typing methods such as multilocus -sequence typing (MLST), classification of the mobile genetic element Staphylococcal cassette chromosome mec ( SCCmec ), spa typing, and DNA microarray for detection of resistance and virulence genes, a number of important clones have been identified. 15 Most of the epidemic MRSA isolates belong to eight major clonal complexes (CCs) including CC1, CC5, CC8, CC22, CC30, CC45, CC59 and CC80. 15 In addition, strains belonging to sporadically distributed clonal complexes such as CC6, CC7, CC9, CC12, CC15, CC20, CC75, CC88, CC93, CC96/ST154, CC97, CC130, CC121, CC152, CC188, CC361, CC395/ST426, CC398, CC509, CC779 and CC913 have been reported. 15,16

21. By inserting the smaller mobile SCC mec  type IV into PVL-positive MSSA, the community-associated MRSA would possess resistance determinants and enhanced toxins, potentially gaining a fitness advantage. Resulting characteristics of community-associated MRSA infections include a lack of hospital-associated risk factors, susceptibility to many non–β-lactam antibiotics, distinct genotypes, and distinct genetic determinants of virulence (e.g., PVL toxin). Gordon and Lowy discuss the molecular epidemiology and virulence associated with community-associated MRSA in this supplement.

22. Changes in the epidemiology of MRSA isolates Beginning in the late 1980s, the MRSA population expanded with the emergence of community isolates. 9,10 clones that have been isolated widely include the HA-MRSA clones ST239/ST241-III-MRSA, ST22-IV-MRSA and the CA- MRSA clones ST80-IV-MRSA, ST30-IV-MRSA, ST772-V-MRSA. On the other hand, clones such as ST59-IV, ST93-IV and ST8-IV (USA300) have restricted geographical spread. 15 The USA300 is the dominant CA-MRSA clone causing infections in North America. 26,27 USA300 have been reported sporadically in some European countries and Australia. 15 It is commonly associated with skin and soft tissue infections, necrotizing pneumonia and endocarditis. 17,18

23. Virulence determinants in MRSA Panton Valentine- leukocidin Panton Valentine leukocidin (PVL) is a toxin composed of two components, LukS -PV and LukF -PV. These two components act together to form pores on the polymorpho- nuclear cells membranes leading to neutrophil lysis. 19 PVL has been reported in HA-MRSA as well as MSSA strains belonging to diverse genetic backgrounds. 20 The toxin is encoded by bacteriophages which explains its carriage in strains belonging to diverse genetic backgrounds. 20 The most PVL- positive S. aureus strains are associated with skin and soft tissue and musculoskeletal infections, some PVL-positive strains also cause invasive infections such as bacteraemia, and necrotizing pneumonia in diverse patient populations. 19,21

24. Arginine catabolic mobile element The arginine catabolic mobile element (ACME) is a novel class of virulence determinants carried on a genomic island which varies in size from 31-kb to 46-kb in staphylococcus. 17 ACME encodes a putative virulence determinant which provides for several immune modulating functions, including resistance to polyamines which serve as a non-specific immune response both on intact skin, and following the inflammatory response in wound healing. It is thought to aid bacterial growth, provide a competitive survival advantage, and enhance the fitness of S. aureus possibly by facilitating colonization and/or hematogenous spread to target organs. 22,23

25. Transmission

26. Transmission MRSA is transmitted from person to person by contaminated hands. Lack of access to hand hygiene products can increase the risk of transmission. Additional risk factors includes, sharing personal products such as shampoo or nail clippers, infrequent showers and hand washing. Another mode of transmission noted within the federal prison system is illicit, unsanitary tattoo practices. In other settings close physical contact, body shaving, turf burns and sharing athletic equipment have been associated with MRSA transmission. Persons with asymptomatic MRSA nasal carriage can shed MRSA resulting in transmission to other persons or contamination of food that may cause toxin mediated acute gastroenteritis.

27. Some MRSA strains, CC398 are readily transmitted with in the host species to which they are adopted. Inhalation of contaminated dust, which can contain large no.of organisms, is thought to be a major route of spread in confinement operations.

28. Identification of MRSA Phenotypic detection methods All isolates of S. aureus are tested by oxacillin disc diffusion, cefoxitin disc diffusion, oxacillin screen agar and latex agglutination tests, and growth on CHROM agar. A standard strain of MSSA (ATCC 29213) and a PCR-positive control strain [ATCC 43300 ( mecA positive)] are used as controls for all methods.

29. Oxacillin disc diffusion method All strains were tested with 1 ug oxacillin discs (Hi-Media) on Mueller–Hinton agar plates. For each strain, a bacterial suspension adjusted to 0.5 McFarland was used. The zone of inhibition was determined after 24 h incubation at 35C. Zone size was interpreted according to CLSI (2008) criteria: susceptible, >13 mm; intermediate, 11–12 mm; and resistant >10 mm.

30. Cefoxitin disc diffusion method All strains are tested with 30 ug cefoxitin discs (Hi-Media) on Mueller–Hinton agar plates. For each strain, a bacterial suspension adjusted to 0.5 McFarland was used. The zone of inhibition was determined after 16–18 h incubation at 35C. Zone size was interpreted according to CLSI (2008) criteria: susceptible, >22 mm; resistant, <21 mm.

31. Oxacillin screen agar test A bacterial inoculum of each strain was made and turbidity was adjusted to 0.5 McFarland. One drop of this suspension was inoculated on Mueller–Hinton agar containing 4 % NaCl and 6 ug oxacillin (Hi-Media). Plates are incubated at 35 C for 24 h. Any strains showing growth on the plate containing oxacillin are considered to be resistant to methicillin.

32. CHROM agar CHROM agar (Hi-Media) is a new chromogenic medium for the identification of MRSA. For each strain, a bacterial suspension adjusted to 0.5 McFarland was used. Subsequently, a swab was dipped in the suspension and streaked onto a CHROM agar plate. The growth of any green colony was considered to be positive, indicating MRSA Latex agglutination test for detection of PBP2a A latex agglutination MRSA screen test (Denka Seiken) was carried out for all strains according to the manufacturer’s instructions.

33. For MRSA, cotrimoxazole (25 mg), erythromycin (15 mg), clindamycin (10 mg), ciprofloxacin (30 mg), netilmicin (30 mg), amikacin (10 mg), linezolid (30 mg), vancomycin (30 mg) and dalfopristin / quinpristin (15 mg) are tested. For MSSA, the same antibiotics as for MRSA are used, as well as ampicillin (10 mg), cephalexin (30 mg) and amoxicillin/ clavulanic acid (30 mg). The Kirby Bauer disc diffusion method is used routinely to detect the sensitivity of all S. aureus isolates and interpretations are made according to CLSI (2008) guidelines.

34. Differences between oxacillin and cefoxitin Oxacillin Cefoxitin Sensitivity is 73.1% Sensitivity is 96.2% Specificity is 96.2% Specificity is 96.2% PPV is 95.0% PPV is 95.0% PPV is 96.2% NPV is 73.8 % NPV is 73.8NPV is 96.2%

35. The superiority of cefoxitin on MRSA detection is because, cefoxitin acts as a strong inducer on mec A gene regulatory system. Cefoxitin is easier to interpret and to read. Oxacillin, which is also the same antibiotic group with methicillin, is cheaper and accessible. Oxacillin replace methicillin which is no longer available commercially in the US and oxacillin is more possible to detect hetero resistant strains.

36. Molecular detection methods MRSA lineages and strains can be identified with molecular tests such as : PFGE (Pulse Field Gel Electrophoresis) MLST (Multi Locus Sequence Typing) MLVA (Multiple Loci VNTR Analysis) SCC (Staphylococcal cassette chromosome) mec typing spa typing.

37. Rapid detection of MRSA Cycling probe amplification or fluorescence technology (crystal MRSA-ID system). Vologene assay using fluorescein conjugated probes. Latex agglutination test . Immunochromatographic strip methods for detection of PBP2a which contains Clear View Exact PBP2a and Binax Now 2a methods for rapid, qualitative detection of PBP2a. Oxoid latex agglutination test for PBP2a.

38. MRSA genes   virulence genes including:  sea (102 bp ),  seb ( 164 bp ) ,  sed ( 278 bp ),   tst ( 326 bp ) ,  eta ( 93 bp ) ,  etb ( 226 bp ) ,  LuKS /F-PV ( 443 bp ) ,  hla ( 209 bp )  and  hld ( 11 bp )   and mec A (147 bp ) in methicillin-resistant  S. aureus. These genes are isolated by using the polymerase chain reaction (PCR) technique.

39. Resistant gene Antimicrobial agent Mechanism of resistance Ars B Arsinite /antimony Active export Bla Z Penicillin Inactivation by a beta-lactamases Cad A Cadmium/zinc Active export Cat Chloramphenicol Inactivation by a chloramphenicol acetyltransferase Erm A MLS Methylation of RNA Fus A Fusidic acid Target alteration in EF-G Fus B Fusidic acid Decreased permeability Mec A Methicillin Production of an altered PBP2 Str Streptomycin Modification by a 6 adenyl transferase Str A Streptomycin Ribosomal alteration Tet A(K) Tetracycline Active export Tet A(M) Tetracycline Ribosomal protection Vat sgA Modification by an acetyl tranferase Vgb sgB Modification by a hydrolase enzyme

40. Epidemiology

41. The frequency of methicillin-resistant  Staphylococcus aureus  (MRSA) infections continues to grow in hospital-associated settings and more recently, in community settings in the United States and globally. The increase in the incidence of infections due to  S. aureus  is partially advances in patient care and also the pathogen ability to adapt a changing environment. The first appearance of methicillin resistance  S. aureus  strains in 1960, has become widespread in hospitals and intensive care units (ICUs). National Nosocomial Infection Surveillance (NNIS) System data demonstrates that, increase in the incidence of nosocomial infections caused by methicillin-resistant  S. aureus  (MRSA) among ICU patients over time. The MRSA now accounts for >60% of  S. aureus  isolates in US hospital ICUs.

42. Hospital associated MRSA The increase in MRSA infections most likely reflects the growing impact of medical interventions, devices, older age of patients. Antibiotic use and overuse probably also contribute to the emergence of resistance. Recent studies demonstrate a continuing increase in MRSA infections in hospitals (CDC investigations). an estimated 1,25,969 hospitalizations annually for  S. aureus  infections in 1999–2000, including bloodstream infections and pneumonia. of the isolates 43.2% were methicillin resistant. Some of the therapies suggesting that β-lactam antibiotics are superior for treatment of these infections.

43. Risk factors for HA-MRSA Being hospitalized.  MRSA remains a concern in hospitals, where it can attack those most vulnerable — older adults and people with weakened immune systems. Having an invasive medical device.  Medical tubing — such as intravenous lines or urinary catheters — can provide a pathway for MRSA to travel into your body. Residing in a long-term care facility.  MRSA is prevalent in nursing homes. Carriers of MRSA have the ability to spread it, even if they're not sick themselves.

44. Community Acquired MRSA The causative bacteria of MW2 strain of community-associated MRSA, which appears to have acquired staphylococcal cassette chromosome (SCC)  mec  type IV, the  S. aureus  pathogenicity island (SaPI3), and the bacteriophage Sa2 in its evolution from MSSA476. in France from 1986 to 1998, children's with pneumonia caused by  S. aureus  strains positive for Panton-Valentine leucocidin (PVL). of 16 children, 12 (75%) presented with an influenza-like illness, and 37.5% died within 48 h after hospital admission. In this series, MRSA isolates were susceptible to non–β-lactam antibiotics and caused clinical disease. Most children presented with skin infections, including cellulitis or abscess. 

45. Treatment

46. Daptomycin It is more active against MRSA/MSSA. MSSA/MRSA MIC’s = 0.5 mcg/ml. Daptomycin is inactivated by calcium in alveolar surfactant fluid and not be used for pneumonia, but useful for septic pulmonary emboli or lung abscess. An initial dose of gentamycin, which increases the intracellular entry or effectiveness of daptomycin when treating with MSSA/MRSA infections .

47. TMP- SMX (same spectrum of ceftriaxone) It is inactivate against most streptococci, it is an excellent antibiotic against MSSA. TMP- SMX is active against CA-MRSA, but it is suboptimal against HA-MRSA. Linezolid Highly effective against major G +ve pathogens. Including, MSSA,MRSA .

48. Tetracyclines MRSA is sensitive to doxycycline invitro. But, it is delayed or incomplete response in in vivo. Clinically, minocycline IV for treatment of MRSA/MSSA is more effective than doxycycline. oral Minocycline to treat serious systemic infections due to MRSA/MSSA. i.e. Osteomyelitis, meningitis. Minocycline is the most cost effective oral anti-MRSA antibiotic. Quinupristin / dalfopristin Highly effective against MRSA/MSSA.

49. Clindamycin It is one of the few antibiotics, able to penetrate or dissolve staphylococcal biofilms. It is effective against CA-MRSA but not HA-MRSA. With CA-MRSA, inducible clindamycin resistance should be suspected. if erythromycin is resistant and clindamycin is sensitive. The D test will conform the clindamycin resistant. Cephalosporins ceftaroline is the only cephalosporin active in vivo against MRSA.

50. Vancomycin vancomycin is one of the major antibiotic for MRSA use. The MRSA is sensitive to vancomycin with MIC being 1 ug /ml. MIC values of vancomycin against MRSA has been increasing worldwide, leading to the emergence of VISA. The combination therapy mainly included the combined regimen of vancomycin and carbapenems with other aminoglycoside drugs, which increase the risk of developing nephrotoxicity and ototoxicity. If combined therapy is needed for MRSA infection, the third generation cephalosporins should be administrated.

51. Combination therapy Combination with vancomycin The Synergetic interactions between the vancomycin and wide variety of beta-lactams are there, but the mechanism is still not clear. The see-saw effect ( if decreased vancomycin susceptibility, which results, decreased transcription of mecA gene and increase the susceptibility of beta-lactams). Combination with daptomycin Daptomycin with beta-lactams, which kills the daptomycin susceptible and daptomycin non-susceptible MRSA. Increases the daptomycin binding to the bacterial cell membrane. Prevents the development of daptomycin resistant strains. It is proved in rabbit model of endocarditis.

52. Prevention

53. Preventing HA-MRSA Preventing HA-MRSA In the hospital, people who are infected or colonized with MRSA often are placed in contact precautions as a measure to prevent the spread of MRSA. Visitors and health care workers caring for people in isolation may be required to wear protective garments and must follow strict hand hygiene procedures. Contaminated surfaces and laundry items should be properly disinfected.

54. Preventing CA-MRSA Wash your hands.  Careful hand washing remains your best defence against germs. Carry a small bottle of hand sanitizer containing at least 62 percent alcohol for times when you don't have access to soap and water. Keep wounds covered.  Keep cuts and abrasions clean and covered with sterile, dry bandages until they heal. The pus from infected sores may contain MRSA, and keeping wounds covered will help prevent the bacteria from spreading. Keep personal items personal.  Avoid sharing personal items such as towels, sheets, razors, clothing and athletic equipment. MRSA spreads on contaminated objects as well as through direct contact. Shower after athletic games or practices.  Shower immediately after each game or practice. Use soap and water. Don't share towels. Sanitize linens.  If you have a cut or sore, wash towels and bed linens in a washing machine set to the hottest water setting (with added bleach, if possible) and dry them in a hot dryer. Wash gym and athletic clothes after each wearing.

55. Antibiotic stewardship Antimicrobial stewardship is defined as “the optimal selection, dosage, and duration of antimicrobial treatment that results in the best clinical outcome for the treatment or prevention of infection, with minimal toxicity to the patient and minimal impact on subsequent resistance”. 24 The goals of antibiotic stewardship are to work with health care practitioners to help each patient receive the most appropriate antimicrobial with the correct dose and duration, to prevent antimicrobial overuse, misuse, abuse and minimize the development of resistance. 24 An added benefit of programs that aim to optimize antibiotic use is that they generally experience cost savings. Because, fewer doses of antibiotic are used and less expensive antibiotics are chosen.

56. Both at the individual patient level and at the community level, antibiotic use changes susceptibility patterns. Patients exposed to antibiotics are at higher risk of becoming colonized or infected by resistant organisms. As hospitalized patients become more complex to treat, the increasing prevalence of antimicrobial resistance in both healthcare and community settings represents a daunting challenge. Antimicrobial stewardship can provide all practitioners with tools to prevent the overuse of valuable resources and help to control the increase in antimicrobial resistance. 24,25

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62. Thank you…..