13 - gram pos superbugs

• Biotyping


  Identification of bacterial species based on biochemical and physiological characteristics
• S. aureus is a key Gram positive bacterium
• Gram positive bacteria


  • Corynebacterium
  • Clostridium
  • Listeria
  • Bacillus
• Gram positive bacteria


  • Optochin sensitive
  • Bile soluble
  • Capsule
  • Optochin resistant
  • Not bile soluble
  • No capsule
  • Bacitracin sensitive
  • Bacitracin resistant
• Streptococcus species


  • S. pneumoniae
  • S. viridans
  • S. mutans
  • S. sanguis
  • S. pyogenes
  • S. agalactiae
• γ-haemolytic


  No haemolysis
• Gram positive cocci


  • Enterococcus
  • E. faecalis
  • E. faecium
• Gram positive cocci


  • Bacilli
  • Cocci
  • Catalase +
  • Catalase -
• Staphylococcus


  Catalase +
• Streptococcus


  Catalase -
• Coagulase


  Converts fibrinogen into fibrin, causing agglutination of rabbit plasma
• Staphylococcus species


  • S. aureus
  • S. epidermis
  • S. saprophyticus
• Identification (Biotyping) of Gram Positive cocci


  1. Visual clue, growth habit

  2. Catalase assay, in slide or tube format
• Catalase


  Protects Staphylococcus from reactive oxygen species
• Catalase assay


  Causes production of gas (bubbles) from peroxide
• MLST (Multilocus Sequence Typing)


  • Technique for typing multiple genetic loci
  • Isolates characterised by deriving DNA sequences of internal fragments of multiple genes
  • Relatively small fragments (approximately 450-500 bp) amplified using specific PCR primers
  • Different sequences present assigned as distinct alleles
  • Allelic profile or sequence type (ST) defines the isolate
• The Pseudomonas aeruginosa MLST Database was developed by Chris Dowson and Barry Curran at the University of Warwick, UK
• MLST-Multi Locus Sequence Typing


  1. Amplify ~450 bp fragments of seven house-keeping genes

  2. Sequence the seven gene fragments

  3. Assign each different sequence at a locus a different allele number

  4. Allele numbers give an allelic profile of the isolate

  5. Compare the allelic profile of the isolate to those of all other isolates in a central database (www.mlst.net)
• Clonal complex


  Group of closely related strains
• High throughput 16S rDNA sequencing


  1. Universal primers used to amplify (by PCR) the rDNA from an isolate

  2. The amplified DNA is sequenced

  3. Sequences are compared
• Treatment should be tailored to the individual, rather than use one standard treatment
• In 1941 all staphylococci were susceptible to penicillin
• By 1951 60% of nosocomial staphylococci were resistant to penicillin
• Methicillin was introduced in 1959 and within two years (1961) methicillin-resistant staphylococci (MRSA) appeared
• MRSA has persisted, but it is waning (good news story)
• Reasons for the reduction in MRSA cases in Europe


  • Reducing the impact of risk factors
  • Improved screening of hospitalised patients
  • Better infection-control practices (isolating patients who have MRSA)
  • Better barrier precautions (doctors wearing sterile gowns)
  • Increasing knowledge of environmental reservoirs and longevity of S.aureus (e.g. on fomites)
  • Improved knowledge of molecular mechanisms of resistance
  • The introduction of the antibiotic Vancomycin
• 92.7% of MRSA deaths in Wales (2010-2014) occurred in NHS hospitals
• Colonization of the nares


  Significant risk factor for subsequent S. aureus infection
• Nasal colonisation by S. aureus


  • 20–30% persistently
  • 30% intermittently
  • 50% never, or rarely
• Nosocomial MRSA risk factors


  • Prolonged hospital stay
  • Treatment with broad spectrum antibiotics
  • Treatment in intensive care or a burns unit
  • Proximity to another patient with MRSA
  • Surgical wounds
  • Infected people from the community entering hospital (patients, visitors, contractors)
• Many patients now become colonized with MRSA in the community (e.g. nursing homes), rather than in hospitals
• Risk factors for Community-Associated MRSA


  • Frequent skin-to skin contact
  • Sports participants
  • Men who have sex with men (MSM)
  • Living in crowded conditions: inmates in prisons, military recruits, children in daycare, elderly in care centres
  • Having or touching cut/grazed skin
  • From fomites
  • Sharing common personal items e.g. towels and razors
  • Touching contaminated surfaces
  • Being HIV positive
  • Getting tattooed
• In 1952 72% of S. aureus isolates were penicillin resistant
• Penicillin resistance mechanism


  Acquisition of β-lactamases that break the β-lactam ring of penicillin
• Methicillin was introduced in the 1960s because it was effective against β-lactamase producing strains of S. aureus
• Low level methicillin resistance


  Hyperproduction of β-lactamase or Modification of the penicillin binding proteins (PBPs) with reduced affinity for methicillin
• High level methicillin resistance


  Acquisition of a completely new PBP - PBP2' - with very low affinity for methicillin, encoded by mecA
• The likely mecA donor is S. sciuri
• Mechanism of action of the mecA product PBP2'


  • The active site of the cell wall cross-linking enzyme PBP can bind methicillin, permanently inactivating the PBP: cell wall cross-linking ceases
  • The mutated active site of PBP2' cannot bind methicillin, so cell-wall cross-linking continues
• Some S. aureus strains are now multiply resistant to a wide range of other antibiotics including aminoglycosides, erythromycin, quinolones and tetracycline