Multi Drug Resistant Organisms - Microbiology Notes PDF

▬▬▬▬ MULTI DRUG RESISTANT ORGANISMS ▬▬▬▬ MDRO – Part 1 Gram-positive organisms: MRSA (ORSA), BORSA, PRSP, VRE & HLAR in enterococci The β-lactamases In order to be effective, β-lactam agents must bind to the penicillin binding proteins (PBPs) in ba...

▬▬▬▬ MULTI DRUG RESISTANT ORGANISMS ▬▬▬▬ MDRO – Part 1 Gram-positive organisms: MRSA (ORSA), BORSA, PRSP, VRE & HLAR in enterococci The β-lactamases In order to be effective, β-lactam agents must bind to the penicillin binding proteins (PBPs) in bacterial cell walls. In their defense, bacteria produce enzymes, called β-lactamases which open up (cleave) the antimicrobial’s beta-lactam ring, altering the structure of the antibiotic. Once the β-lactam ring is modified, the antimicrobial cannot bind to the PBPs, and the antimicrobial is rendered ineffective. hundreds (well over 500) of β-lactamase enzymes have been documented o β-lactamase enzymes have been documented against every β-lactam drug most β-lactamases inactivate either penicillins (called penicillinases) or cephalosporins (cefinases) – and some can inactivate both drug classes. multiple β-lactamases are possible in the same strain of bacteria detection of β-lactamases can be difficult: the chromogenic beta-lactamase test (Nitrocefin is the substrate) only determines if narrow spectrum agents (e.g., penicillin and ampicillin) are affected, and in only a few species. This direct testing is not always useful – it doesn’t tell us which β-lactamase is produced. many β-lactamases are found only by through looking at AST patterns in specific antimicrobials – eg., to find the extended spectrum beta-lactamases (ESBL’s) in the Enterobacterales, we look at the resistance/susceptibility patterns of the first and third generation cephalosporins. hyper-production of β-lactamase can knock out a normally effective β-lactam drug. Hyper- production = where an organism starts to produce more beta-lactamase in response to increasing concentrations of a beta-lactam agent. o e.g. hyper-β-lactamase producing strains of S. aureus can show resistance to oxacillin, which is considered a penicillinase-resistant β-lactam agent some β-lactamases are constitutive – meaning they are always produced others are inducible - β-lactamase production is initiated (or turned on) when an organism that has a β-lactamase gene is exposed to a β-lactam agent – for example, when the patient takes the drug. Conversely, production is turned off when no antibiotic is present in or around the cell β-lactamase Inhibitors: β-lactam agents are combined with β-lactamases inhibitor drugs: clavulanic acid, tazobactam and sulbactam are the earliest ones developed. o newer drugs: avibactam, relabactam and vaborbactam o the β-lactamase inhibitor binds to the β-lactamase enzyme preventing it from cleaving the β-lactam ring. β-lactamase inhibitors have no activity against the organism itself o combination drugs now seen in our AST panels:  amoxicillin/clavulanic acid (Augmentin) & ticarcillin/clavulanic acid (Timentin)  piperacillin/tazobactam (Tazocin or Zosyn) & ceftolozane/tazobactam  ampicillin/sulbactam  ceftazidime-avibactam  imipenem-relabactam  meropenem-vaborbactam MLSC 3309 – Clinical Microbiology 2 Classification of beta-lactamases Ambler system (1980) is based on molecular structure and amino acid sequence A, C, and D Class β-lactamases utilize serine for β- lactam hydrolysis Class B metallo- β-lactamases require divalent zinc ions for substrate hydrolysis Bush system (1995) is based on the substrates that a β-lactamase hydrolyses and which compounds can inactivate it o specifically: beta-lactamase inhibitor drugs, EDTA, azthreonam or cloxacillin takes into account substrate and inhibitor profiles in an attempt to group the enzymes in ways that can be correlated with their phenotype in clinical isolates. these major groupings generally correlate with the more broadly based molecular classifications. o Group 1 (Ambler class C) are cephalosporinases o Group 2 (Ambler classes A and D), which is divided further into 8 subgroups includes:  broad-spectrum, inhibitor-resistant β-lactamases  extended-spectrum β-lactamases (ESBLs)  serine carbapenemases o Group 3 (Ambler class B) are the metallo-β-lactamases. o Group 4 is yet undetermined Fall 2024 2  MLSC 3309 – Clinical Microbiology 2 Gram Positive β-lactamases are exoenzymes: they are secreted by the organism and diffuse into the external medium most are in molecular class A or Group 2a and are generally inhibited by clavulanic acid Beta-lactamases in Staphylococci Penicillinase: by simple definition, staphylococci that are “S” to penicillin should be susceptible to other penicillins, cephalosporins and carbapenems the first penicillin resistant strain of staphylococci was reported in 1944  just 2 yrs after the introduction of the drug  this β-lactamase is called penicillinase. today approx. 90% of staphylococci produce penicillinase - are therefore considered “R” to penicillin. these strains are “R” to the penicillinase-susceptible penicillins: penicillin, amoxicillin, ampicillin and piperacillin but, are generally “S” to the penicillinase-resistant penicillins: oxacillin, cloxacillin & methicillin. (these are also called penicillinase-stable penicillins) and are also “S” to  the cephalosporins  the β-lactam/β-lactam inhibitor combination drugs: e.g. amoxicillin/clavulanate,  the relevant cephems and carbapenems the penicillinase-resistant penicillins are NOT affected by normal levels of beta-lactamase Inducible β-lactamases:  if a strain of staphylococci tests “S” to penicillin, we must test for inducible β-lactamase  exposing the strain to a beta-lactam agent first (penicillin, oxacillin or cefoxitin), and then performing a direct β-lactamases test (test method below): Direct β-lactamase testing of staphylococci (for both S. aureus & CoNS) Required: growth from the edge of a zone of inhibition around either a penicillin or cefoxitin disc OR from the ellipse edge of a penicillin or oxacillin MIC strip Culture must be fresh (overnight incubation) Apply the test organism to a moistened nitrocefin test disc or strip Incubate at room temperature (RT), following the manufacturer’s instructions for timing – most recommend a 1 hour incubation period for staphylococci Observe for the development of an orange/red colour This test is also available in liquid form – trade name is Cefinase Penicillin Edge-Zone Test (for strains of S. aureus ‘S’ to penicillin) Only for strains of S. aureus with penicillin zone sizes ≥ 29mm or MIC’s ≤ 0.12 ug/ml [test “s” to penicillin] This test is NOT applicable to strains that test “R” to penicillin Method: Mueller Hinton Agar, #0.5 McFarland, 10 unit Penicillin disc incubate in ambient air @ 35±2°C, 16-18 hrs result: sharp zone edge (‘cliff’) = β-lactamase POSITIVE result: fuzzy zone edge (‘beach’) = β-lactamase NEGATIVE The first paper on this was published in Oct 1982 in The Journal of Clinical Microbiology. These researchers had 100% correlation for S. aureus and 97% for CoNS. CLSI updated their Tables and added this test to the M100 publication in 2013, over 30 yrs later! Fall 2024 3 MLSC 3309 – Clinical Microbiology 2 Beta-lactamases in Enterococci: all enterococci exhibit decreased susceptibility to penicillin and ampicillin, as well as high level resistance to semi-synthetic penicillins and most cephalosporins. resistance to ampicillin and penicillin due to β-lactamase production cannot be reliably predicted by AST: we must test specifically for it using the direct nitrocefin test. the nitrocefin method as described previously, but prior exposure to the beta lactam agent is NOT necessary (this is not an inducible beta-lactamase in enterococci)  incubation is just 5 minutes, [a few manufacturers say 15 minutes]  in any case, it’s less incubation time than staphylococci if determined to be a non-β-lactamase producer, then the isolate is predictably susceptible to ampicillin, amoxicillin, and piperacillin. all labs will test isolates from critical specimens: blood cultures, body fluids and CSF’s  some labs test all enterococci isolates only reported if the result is positive remember, they are very rare The Gram-negative β-lactamases these β-lactamases exist in the periplasmic space of the cell wall o they become concentrated in that space and are always available they vary markedly: o may have penicillinase and/or cephalosporinase activity o may or may not be bound by inhibitors o may be constitutive or can be inducible o may be chromosomal or plasmid encoded detection is difficult – many cannot be detected by standard AST methods. For clues, we can look at the resistance/susceptibility patterns in the antibiogram. for the above reasons, direct β-lactamase testing is rarely done on Gram-negative organisms o the exceptions:  Haemophilus influenzae  Neisseria gonorrhoeae  Moraxella catarhalis  and some Bacteroides species o same test procedure as described on Pg 3 o no previous exposure to a beta-lactam agent is required o BUT the reaction (colour change) is almost immediate  why this difference from Gram- positive organisms? beta- lactamases produced by gram- negative organisms are held within the cell, they are not secreted and therefore open the beta-lactam ring of the Nitrocefin very quickly. Gram-negative cell wall Fall 2024 4 MLSC 3309 – Clinical Microbiology 2 Altered Target Resistance: Some resistant bacteria evade antimicrobials by reprogramming or camouflaging the antimicrobial’s target. This strategy has been observed these areas: staphylococci against methicillin and other beta-lactam agents, i.e., MRSA (ORSA) o ORSA (Oxacillin Resistant S. aureus) is the more accurate term inducible resistance to clindamycin in staphylococci, BH streptococci and S. pneumoniae enterococci against vancomycin (VRE) vancomycin resistance in S. aureus (VISA & VRSA) increasing resistance to penicillin in Streptococcus pneumoniae (PRSP) A Quick Review of Penicillin-Binding Proteins (PBPs) penicillin-binding proteins (PBPs) are a group of proteins in bacterial cell walls that are characterized by their affinity for and binding of penicillin. are a normal constituent in most bacteria o the name just reflects the way by which the protein was discovered. PBPs are members of a subgroup of enzymes called transpeptidases o they are used in the building of the bacterial peptidoglycan layer of the cell wall o are bound to the inner plasma membrane each PBP has a different function in building the cell wall. the loss of some PBPs is bactericidal (lethal), while the loss of others is less critical. each organism possesses several PBPs. They are numbered according to molecular weight (MW) o the smallest PBP number has the highest MW. o tPBP1’ in one species is different from ‘PBP1’ in another species PBP’s can be altered in one step, or it may take several: o in MRSA, the mecA gene alters PBP2 to PB2a in a one-step process  (Note: you’ll also see PBP2a noted as PBP2') o in S. pneumoniae, high level resistance to beta-lactam agents requires several successive alterations in essential PBPs. altered PBPs are rare in enteric Gram-negative bacilli and Pseudomonas resistance to β-lactams in Haemophilus influenzae, Neisseria gonorrhoeae and Neisseria meningitidis is usually by β-lactamase, but in rare cases is due to altered PBP. o one example: BLNAR Haemophilus influenzae = beta-lactamase negative ampicillin resistant. The direct beta-latamase test is negative, but the testing of ampicillin = I or R. This is due to an amino acid substitutions in PBP3 most beta-lactam agents have some affinity for both gram positive and gram negative organism, exception: aztreonam is the only β-lactam drug that has no affinity for Gram-positive PBPs Fall 2024 5  MLSC 3309 – Clinical Microbiology 2 Methicillin (Oxacillin) Resistant S. aureus – MRSA (ORSA) Methicillin (oxacillin) resistant S. aureus are defined as strains that have MICs to oxacillin ≥ 4 ug/mL. another way to say this: strains that have MICs to oxacillin > 2 ug/mL. (4, 8, 16, 32 and so on) most true MRSA’s (ORSA) actually have MICs well above 16 ug/mL. their resistance is due to an altered penicillin binding protein (PBP): o PBP2 which is altered to PNP2a (a.k.a PBP2’). the mecA gene is responsible for coding for this protein. Border-line oxacillin resistant S. aureus or BORSA’s MICs from 4-16 ug/mL DO NOT carry the mecA gene. resistance is due to the hyper-production of beta-lactamase. strains test: o “R” to oxacillin, but o “S” to amoxicillin/clavulanic acid. Research: OX disc testing on MHA @ 30C can detect BORSA’s (Zones 10-13 mm)* Modified S. aureus (MODSA) – emerging group. Similar to MRSA strains, MODSA do not become susceptible to penicillinase resistant penicillins (PRPs) after the addition of a beta-lactamase inhibitor. These modifications typically involve PBP3 and/or PBP4, and result from the selective pressure of beta-lactam antibiotics.** the first step in determining whether a strain of S. aureus is a true MRSA (ORSA), is to first confirm that the organism is indeed Staphylococcus aureus o our usual ID battery of colony morphology, smear, catalase, slide coagulase and tube coagulase or Latex coagulase testing (Staphaurex, etc.) is not acceptable for confirming the ID of S. aureus when we suspect it to be an MRSA (ORSA) o a full biochemical/phenotypic ID is required,  Microscan panel, Vitek or Phoenix card or API strip are all acceptable  Maldi ID acceptable, but is usually confirmed with TC o other species of staphylococci can be TC POS and R to oxacillin – e.g., S. intermedius latex testing kits specific for PBP2a have been developed and are in use in most clinical labs o are rapid and can be done from primary plates o on the first day that S. aureus is suspected, are a quick screen for MRSA (ORSA) o these can also be used with coagulase negative staphylococci o if POS, strain has PBP2a = mecA gene Antimicrobial Susceptibility Testing: determining an isolate’s susceptibility to oxacillin is done for both patient treatment and Infection Control reasons o hospitals are always looking for MRSA (ORSA) clusters and outbreaks all clinically significant isolates of S.aureus require testing for resistance to oxacillin o test oxacillin OR test just cefoxitin (a surrogate drug for oxacillin) o oxacillin MIC from instrument panels is not valid (CLSI) – time & temperature issues as most MRSA strains are R due to mecA mediated resistance, determining whether or not a strain has the mecA gene is critical (remember that resistance is not always expressed) the two most accurate methods for predicting mecA mediated resistance to oxacillin are: o testing for the mecA gene by PCR & o using a latex kit to detect the PBP2a protein products of the mecA gene Fall 2024 6 MLSC 3309 – Clinical Microbiology 2 MRSA strains are hetero-resistant: have both “S” and “R” subpopulations o heteroresistance issue: the resistant subpopulation is slower growing o this is why we have to use the direct inoculum method to set up all AST for staphylococci, and cannot use the growth/log phase method. three AST methods are available to determine resistance to oxacillin in strains of S. aureus: 1. broth dilution, both macro and micro methods 2. gradient diffusion - Etest 3. agar dilution method – OX screening medium Method 1: Broth Dilution disc diffusion testing is not reliable for oxacillin – has been for about a decade therefore an MIC method is required broth dilution: o CAMHB (cation adjusted Mueller Hinton broth) with 2% NaCl o incubate: ambient, 33-35°C, full 24 hrs, o reminder 1: temperatures above 35C can inhibit the resistant sub-population o reminder 2: drugs other than oxacillin can be read at 16 – 20 hrs, incubation temperature 35 ± 2°C and extra NaCl is not required micro-broth results from automated instruments are not valid – testing must be done off-line. Why? incubation times & temperature. Method 2: Gradient Diffusion MIC strips – Etest (Biomerieux) or MICEvaluator (Oxoid) MHA with 2% salt (the higher salt content pushes expression of the mecA gene without affecting the susceptible sub-population) #0.5 McFarland, colony suspension method only – never use the growth method. o Note: a #1.0 McFarland is used in laboratories that use EUCAST standards. CLSI will most likely move to this at some point in the coming years incubate: ambient, 33-35°C, for a full 24 hrs – note the slightly lower temperature a – oxacillin disc diffusion (note tiny colonies in zone) – disc diffusion is no longer CLSI approved b – oxacillin Etest MIC strip: shows heteroresistance: some cells test as R, while others test S c– oxacillin Etest MIC strip: a fully resistant strain Fall 2024 7 MLSC 3309 – Clinical Microbiology 2 Method 3: Oxacillin resistance screening – agar dilution this method screens for strains of S aureus that have an MIC above 4 ug/mL ( ≥ 8) o Note: CLSI breakpoints:  S. aureus: ≤ 2 is ‘S’ ≥ 4 is ‘R’ [no ‘I’ breakpoints]  CoNS: ≤ 1 is ‘S’ ≥ 1 is ‘R’ [no ‘I’ breakpoints] procedure: o a #0.5 McFarland standard colony suspension inoculum o spotted onto a MHA plate with 4% NaCl and 6ug/mL of oxacillin o incubation is at 33-35°C for a full 24 hours (note temperature & time) interpretation: o any growth (even 1 colony): sub to a BAP and test conventionally – MIC strip, etc… o no growth: oxacillin “S” detection of MRSA is optimized with increased salt and the lower temperature o slightly lower temp helps us find R subpopulations o MRSA strains grow well at the higher salt concentration  it pushes the expression of the mecA gene  non-MRSA strains are also slowed down a bit by the high salt concentrations this method is used for S. aureus only used primarily by larger volume labs testing many strains of S. aureus in a day o to avoid having to do an MIC strip on each strain - which is expensive. some recent reports indicate that up to 6% of MRSA are missed with this screening o strains with mic’s of 4 ug/mL are not detected o suggestion has been made to move to 2 ug/mL screen  but this would also pick up many BORSA’s – so confuses the issue o remember: most true MRSA have MIC’s above 16 ug/mL Agar dilution plates after 24 hrs incubation: Left MHA w/4% NaCl only (Growth Controls) – 52 strains tested on one plate (too many IMO!) Right MHA w/4% NaCl & 6ug/mL oxacillin: 4 strains are R to oxacillin, 48 are S Fall 2024 8 MLSC 3309 – Clinical Microbiology 2 Treatment of infections with MRSA since the altered PBP affects the ability of all beta-lactam agents to bind to staphylococcal cells, all penicillins, cephalosporins, carbapenems and beta-lactam/beta-lactamase inhibitor combinations should be reported as resistant, regardless of the in vitro testing results most MRSAs are multi-resistant, e.g. resistant to macrolides, quinolones and aminoglycosides community-acquired MRSA (CA-MRSA) were at one time fairly susceptible to other classes of antibiotics but are becoming as resistant as the hospital strains o notably isolates possessing genes for the Panton-Valentine leukocidins (PVL) toxin vancomycin and teicoplanin are most commonly used, IV only ceftaroline – a new cephalosporin subclass (a Test/Report Group B drug or 2nd line for us) Vancomycin has long been the drug of choice to treat MRSA infections its efficacy is now being questioned: o slow bactericidal activity o emerging resistant strains o possible ‘MIC creep’ among susceptible strains o tissue penetration is variable (affected by inflammation); limited penetration into bone, lung and CNS Teicoplanin a glycopeptide, similar activity as vancomycin not approved for use by the FDA or Health Canada – used extensively in Europe effective against MRSA fewer side effects than vancomycin – less nephrotoxicity, less ‘red man’ syndrome Ceftaroline CLSI categorizes this drug as a cephem a new subclass of cephalosporin: cephalosporins with anti-MRSA activity has a high affinity for PBP2, including PBP2a side effects: diarrhea, nausea and headache not yet approved for use in Canada, USA & Europe only (2020) Other treatment options the tetracyclines and SXT are seen as viable alternatives for treating MRSA infections, particularly if it’s community-acquired and non-invasive clindamycin , although not specifically approved for MRSA, is used extensively as it is efficient with S. aureus rifampin is also often used, but due to rapid development of resistance, is not recommended for monotherapy newer drugs have been developed to treat serious infections: o linezolid, daptomycin and tigecycline: costly and have serious side effects Linezolid (Zyvoxam) an Oxazolidinone, inhibits protein synthesis used for MRSA, VISA, E. faecium VRE, and PRSP ineffective for Gram-negative infections, mixed infections toxic effects: bone marrow suppression Note - in 2007 the FDA issued an alert that linezolid should NOT be used for catheter-related infections; a Feb. 2009 article in IDSA News (Infectious Disease Society of America News) states that studies to date are still insufficient to lift this warning and advises that vancomycin continue to be the first-line choice Fall 2024 9 MLSC 3309 – Clinical Microbiology 2 Daptomycin a lipopeptide, it disrupts the Gram-positive organism’s cell membrane by the formation of transmembrane channels cannot permeate the outer membrane of Gram-negative organisms rapid – bactericidal activity against o staphylococci (MRSA, VISA, and VRSA) o enterococci, including VRE o streptococci including drug-resistant S. pneumoniae o most other aerobic and anaerobic Gram-positive bacteria toxic effects: myopathies (affects muscle function) Tigecycline (Tygacil) a new class = glycylcyclines, similar to the tetracyclines received FDA approval in June 2005 bacteriostatic, inhibits protein synthesis – binds to the 30s ribosomal subunit effective against both Gram positive, gram negative organisms and anaerobes MRSA, Stenotrophomonas maltophilia, Haemophilus influenzae, N. gonorrhoeae, and multi- drug resistant strains of Acinetobacter baumanii has no activity against Pseudomonas aeruginosa or Proteus species IV only side effects: diarrheae, nausea, vomiting not for children or pregnant women: affects teeth and bones as do the tetracyclines Screening Patients for MRSA carriage done to detect carriage = colonization in the absence of infection done for purposes of Infection Control and possibly for decontamination patients are screened directly for carriage of MRSA screened patients include:  in-patients transferred from another hospital  patients in ward outbreaks  patients with a previous admission to an out-of-Canada hospital MRSA is increasingly seen as community-acquired (CA-MRSA) the big question: should we screen all admitted patients? the move in many hospitals is to pre-screen all elective surgery patients about one week before admission = if POS  treatment with mupirocin gel (applied topically for 4 days) light therapy becoming the norm: methylene blue solution + UV light critical surgeries are the priority for now:  open heart and other cardiac  thoracic  orthopedic real-time PCR is available for ER admissions specific anatomical sites are screened:  anterior nares: if both Lt and Rt nares are swabbed, inoculate both swabs on to one plate.  axillae (as above “bilateral”)  wounds, drain sites, etc. o especially if the patient was previous MRSA positive Fall 2024 10 MLSC 3309 – Clinical Microbiology 2 Screening Procedures traditional: mannitol salt agar o contains 2mg/L oxacillin (MSAO) o mannitol fermenters: yellow colonies = possible MRSA o CoNS produce white or pink colonies o note: there are rare mannitol fermenting CNS o further workup is required to confirm ID o BAP often inoculated along w/MSAO common today: MRSA chromogenic agar o inoculate chromogenic agar directly with specimen swab, streak for isolation o incubate at 35ºC, check daily for up to 72 hours o some formulas are read at 24 - 28hrs – no re-incubation required o the detection colour varies by manufacturer o use antimicrobials & chromogenic substrates  formulas are proprietary  MRSA Smart Agar (Biomerieux) MRSA Select II  (BioRad) Fall 2024 11 MLSC 3309 – Clinical Microbiology 2 Example of the workup of suspect MRSA colonies from screening agars growth on CHROM agar → smear, catalase, slide coag, tube coagulase w/BAPpp if TC + at 4hrs notify Infection Control (asap if this is a new patient) if TC – from the CHROM agar, it must be repeated from the BAPpp BAP is also used for susceptibility testing o full ID panel is also required – to verify the identification perform susceptibility testing (AST) mecA molecular testing may be done at this point to confirm MRSA o or latex PBP2a kit AST testing may also be performed to test the decontamination drugs o the efficacy of these antibiotics is debatable o patients will re-colonize o typical agents: rifampin, mupirocin (e.g. Bactroban™ applied to the skin or nares), fusidic acid o in Jan 2009, CLSI added high-level mupirocin resistance testing to AST guidelines for S. aureus (uses a 200-μg disk). A few notes on other Staphylococcus spp. and screening tests: Staphylococcus lugdenensis  CLSI Tables: is included in the S. aureus Group  cefoxitin: follow the procedures for S. aureus  use MHA w/2% NaCl for oxacillin testing – all staphylococci  but, the agar dilution screen method (MHA w/ 4% NaCl and 6 ug/mL oxacillin) that is used to detect oxacillin resistance applies to S. aureus only CoNS (coagulase-negative staphylococci)  procedures for CoNS do not include S. lugdenensis  testing for oxacillin resistance for mecA-mediated oxacillin resistance in the CoNS is only done by cefoxitin disk diffusion. see CLSI Table 3E – broth dilution only lists S. aureus & S. lugdunensis but, incubation time and zone diameters for other CoNS differ from the S. aureus Group o full 24 hrs incubation is still required  use MHA w/2% NaCl for oxacillin testing – used for all staphylococci  but, the agar dilution screen method (MHA w/ 4% NaCl and 6 ug/mL oxacillin) used to detect oxacillin resistance applies to S. aureus only WHY? CoNS breakpoints for oxacillin are: S 20 mm), no other beta-lactams need to be tested. o if the screening result is < 20 mm, all beta-lactam agents are tested by MIC method. others that may be tested and reported: and vancomycin, as well as erythromycin, SXT, tetracycline, clindamycin and even cefepime (a fourth gen cephalosporin) and telithromycin ( the first ketolide antibiotic, similar to the macrolides). for CSF isolates: only penicillin, cefotaxime, ceftriaxone, meropenem & vancomycin should be reported o oxacillin disc screening method is never done for isolates from CSF → MIC right away o most clinical labs treat blood culture and body fluid isolates the same as CSF isolates o both the MIC value and the interpretation should be reported (not just S, I or R) o for example: for an MIC of 0.32 μg/mL, the report might read something like this: “Penicillin MIC = 0.32 μg/mL Interpretation: Intermediate (I) resistance” For isolates of S. pneumoniae isolated from CSF, only the meningitis breakpoints need be reported. For isolates recovered from non-CSF isolates, meningitis, non-meningitis plus the oral Pen G results are all reported. This is due to concerns over these infections seeding to the meninges. Fall 2024 14 MLSC 3309 – Clinical Microbiology 2 Vancomycin-Binding Proteins vancomycin binds to the pentapeptide peptidoglycan precursor molecule as it exists in the cytoplasmic membrane. When this occurs, transpeptidation, the cross linking needed to build the cell wall, cannot occur. A normally functioning cell wall cannot be built. the vancomycin resistance gene cluster in enterococci codes for the precursor proteins that modify the vancomycin target binding site from D-ala:D-ala to D-ala:D-lac.. The altered pentapeptide structure is functional for the organism, but will not bind vancomycin. This makes the drug ineffective the most important genes involved in vancomycin resistance are vanA, vanB and vanC. o vanA - altered cell wall precursor molecules confer high-level vancomycin resistance in Enterococcus spp. with the vanA gene - they don’t bind vancomycin OR teicoplanin.  the first VRE isolates were found in the US and Canada in the 1980’s  in 2014 – there were 294 reported cases in Canada. o the vanB cluster produces a carboxypeptidase that acts on the pentapeptide and produces similar changes, but confers only intermediate resistance to vancomycin, and does not confer any resistance to teicoplanin o vanC codes for an intrinsic, low level resistance that is not transfereable. o vanA and vanB both code for a high level resistance that is transferable within the species and across genera. VISA: vancomycin intermediate S. aureus o MIC’s of 8 – 16 ug/mL o first isolated in Japan in 1997, in the US in 2020  Michigan, a patient on peritoneal dialysis: a S. aureus that was both MRSA & VISA. o 17 confirmed US cases as of 2007, 1 case confirmed in Canada to date o mechanism of resistance is not completely understood. VRSA: vancomycin resistant S. aureus. o MIC > 32 ug/mL o resistance coded by vanA o VRSA was first documented in the US in 2002 o 13 confirmed US cases to 2014, no cases in Canada to 2014 Watching for this resistance in S. aureus is the reason we are now doing an MIC method for testing strains of S. aureus to vancomycin. Fall 2024 15 MLSC 3309 – Clinical Microbiology 2 Vancomycin Resistant Enterococci (VRE) The enterococci, as a group, tend to be resistant to a number of antibiotics: cephalosporins, aminoglycosides, clindamycin, oxacillin and trimethoprim-sulfamethoxazole, regardless of testing results. enterococci are not a major cause of infection in the outpatient population are an increasing cause of nosocomial infections – immunocompromised patients o urinary tract infections, endocarditis, wound and intra-abdominal infections o most infections are endogenous but cross infection between patients can occur vancomycin is one of the few drugs that can be used to treat these infections. since their first appearance in 1988, the vancomycin-resistant enterococci (VRE) have emerged worldwide and are becoming an increasing problem in clinical settings. Acquired high-level vancomycin resistance genes are carried on plasmids, which make them easy to spread. seen most commonly in E. faecalis and E. faecium o but can occur in other species (rare). the genes of concern: o vanA gene, which produces an MIC of >128 mg/L o vanB, with MIC’s of 64-128 mg/L the vanA gene has been transferred to S. aureus (VRSA). Intrinsic low-level resistance to vancomycin is seen in the species E. casseliflavus, E. mundtii and E.gallinarum. gene responsible for low level, intrinsic resistance is vanC, o typically produces MICs of 2-16 mg/L. VanD, vanE and vanG have only recently been characterized. Enterococci of significance it is important to distinguish between the Enterococcus spp. for infection control purposes and to guide treatment Enterococcus faecalis and E. faecium are the most commonly isolated o are the most likely to carry the vanA and vanB genes intrinsically resistant enterococci are usually not clinically significant o have not been implicated in outbreaks o vanC genes are not transferable between organisms full ID to the species is not necessary if preliminary tests point to one of the intrinsically resistant enterococci the following chart provides test reactions that are used to screen vancomycin resistant enterococci E. faecalis E. faecium E. gallinarum E. casseliflavus E. mundtii E. raffinosis Motility - - + + - - Pigment - - - + + - Sorbitol ferm - - - - - + Arabinose ferm - + + + + + Pyruvate ferm + - - V - + 2 hr xylose - - + Fall 2024 16 MLSC 3309 – Clinical Microbiology 2 use a a swab to detect the yellow-orange pigment of E. casseliflavus and E. mundtii o if pigment test is NEG, these two are ruled out o if pigment is POS = not a clinically significant VRE pigment NEG  proceed to the 2 hour xylose test: o if POS = E gallinarum, o if NEG = proceed to other tests or full ID motility tests may be done by one of : o motility media (overnight incubation) or o direct microscopy = fresh growth: after 2 hr incubation at 30°C all clinically significant isolates of Enterococcus spp. are screened for vancomycin resistance disk diffusion is still used – but we read vancomycin with transmitted light! o and a full 24 hr incubation is required vancomycin resistance is not be easily detected in all automated systems o incubation time required for microbroth dilution is a full 24 hrs – but most instruments read and discard the panel at 16hrs [or even earlier].  16 – 20 hrs for the other drugs agar dilution screening is used: 6 µg/mL vancomycin in brain heart infusion (BHI) agar o a #0.5 McFarland suspension is spotted ont a BHIA w/6ug/mL vancomycin o read only after a full 24 hours of incubation. o all positive screen results must be confirmed by an MIC method o in serious infections/critical sires, beta-lactamase and synergy testing is also performed. o why 6 µg/mL? Breakpoint for S is ≤ 4 µg/mL o why BHI and not MHA? a few reasons: o studies conducted in the ‘90’s showed that most strains of E. galiinarum did not grow on the MHA, so were missed  MHA was often more difficult to read – enterococci are very transluscent on MHA, and incubation to 48hrs was often required.  on BHI, enterococcus produces more ‘opaque’ growth at 24hrs - easier to read.  paper published in 1994 in the Journal of Clinical Microbiology Screening Patients for VRE Carriage purpose: to detect VRE carriage = colonization in the absence of infection done for Infection Control purposes only o patients identified VRE POS by screening are considered colonized, NOT infected o why screen?  VRE can contaminate the environment around a patient contamination increases with diarrheae  can survive on hard surfaces for days even weeks commodes, shower chairs, patient lockers, bedtables, handrails, etc. o hospital wide screening is not recommended  selective admission and/or interval screening of high-risk inpatient groups only Fall 2024 17 MLSC 3309 – Clinical Microbiology 2 risk factors for VRE carriage: o severe underlying disease (e.g., long term dialysis patients) o recent hospitalization  particularly admission to an intensive care unit o prolonged or broad spectrum antibiotic use – particularly vancomycin o long duration hospital stay o indwelling urinary catheter o close contact with a VRE carrier – caregiver handling soiled diapers, linens, etc patient factors that increase the risk of transmission of VRE o diarrhea or uncontained fecal incontinence o discharging wounds that cannot be contained by the dressings o enterostomy o poor personal hygiene (compliance or inability to manage) risk factors for VRE infection in a health care setting o patients with severe neutropenia o patients undergoing a solid organ transplant o patients requiring admission to ICU’s or neonatal units o presence of an indwelling urinary catheter anatomical sites screened : o stool, perineum/rectum: most common o neck & groin folds in neonates o less common sites: urine, open wounds, drain sites procedures 1. BEAV: Bile Esculin Azide Agar with 6 mg/L Vancomycin bile inhibits Gram-positives other than enterococci esculin differentiates azide (sodium azide) inhibits Gram-negative organisms vancomycin inhibits gram positives & selects for VRE incubate in ambient air at 35ºC examine daily for 72 hours VRE on BEAV: brown-black colonies with a dark halo work up suspect colonies : o cs/ gpc, catalase (-) = possible enterococci → PYR  if PYR (+) → speciate  sub to BAP for other tests including AST 2. VRE Selective Broth: a selective enrichment broth inoculated with ‘pea sized’ stool sample or rectal swab incubate no more than 18hrs, look for evidence of back/grey growth then subculture to VRE selective agar a. formulas can be customized: i. vancomycin only ii. vancomycin + added meropenem: suppresses gram negatives and E. gallinarium iii. vancomycin + added gentamicin: added recovery of HLAR enterococci Perform further susceptibility testing (and or vanA/vanB PCR testing) o Vancomycin resistant: MIC ≥ 32 μg/mL 3. a variety of Chromagar plates are commercially available: o most identify E faecalis & E. faecium o a few also show E. galinarium Fall 2024 18 MLSC 3309 – Clinical Microbiology 2 Example of Workflow for VRE Detection from Screening Cultures (from the Guidelines for the Testing and Reporting of Antimicrobial Susceptibilities of VRE) Fall 2024 19 MLSC 3309 – Clinical Microbiology 2 Treatment of VRE: Patients who test positive for VRE, are often only colonized and do not have an active infection. Colonization is not usually treated, but if there is an infection is present, treatment is required. Enterococci that are vancomycin resistant may still be susceptible to ampicillin. linezolid & daptomycin: see the MRSA notes notes on quinupristin-dalfopristin (Synercid) are below. there have been enterococci reported that are resistant to all known antibiotics.  Quinupristin-dalfopristin (Synercid) a Streptogramin, interferes with protein synthesis  suitable for all Gram-positives  good activity against E. faecium VRE (-cidal)  only-static against others – (so, not suitable for E. faecalis VRE)  not considered a good choice for MRSA because of loss of efficacy if MRSA is macrolide-lincosamide-streptogram resistant constitutively  Teicoplanin  a glycopeptides  is a somewhat less toxic alternative to vancomycin  suitable for serious infections due to vanB E. faecalis VRE and MRSA FYI: a synergy between oxacillin and vancomycin is being researched in strains of S. aureus that are MRSA and exhibit high level vancomycin resistance. This has been documented in strains of MRSA recovered from patients who are also VRE carriers. Thought to be due to lateral transmission of the gene clusters for VRE to these staphylococci. See below: Fall 2024 20 MLSC 3309 – Clinical Microbiology 2 High Level Aminoglycoside Resistance (HLAR) in Enterococci Enterococci are characteristically resistant to a wide variety of antimicrobial agents making single-drug therapy often ineffective. All enterococci naturally have some low-level resistance to aminoglycosides o this invalidates AST with the usual concentrations of antimicrobial agents Many isolates of E. faecalis and E. faecium have acquired high-level resistance to one or more of the aminoglycosides. Systemic infections, such as endocarditis, are usually treated with a combination of two antimicrobial agents: o one specific for action against the cell wall, such as a beta-lactam or a glycopeptide (i.e., penicillin, ampicillin or vancomycin) and o an aminoglycoside, which inhibits bacterial protein synthesis (i.e., gentamicin or streptomycin). o these agents act synergistically to enhance killing of the bacteria, since the aminoglycoside has increased uptake into the cell after the cell wall is damaged by the beta-lactam agent. When an enterococcal strain has high-level resistance to aminoglycosides (HLAR), synergism will not occur, and combination therapy with a beta-lactam drug will not be effective. Therefore, it’s important to detect the presence of this HLAR in order to predict this synergy. Strains that show HLR to gentamicin, the most commonly used and best aminoglycoside against enterococci, possess one or more aminoglycoside-modifying enzymes. o these enzymes may make them resistant to one or more of a variety of other aminoglycosides, including tobramycin, netilmicin, and amikacin, but not streptomycin. Other HLAR enzymes are active against streptomycin, but not gentamicin. Thus, testing both gentamicin and streptomycin provides information on the two most active of the aminoglycosides. If a strain has high-level resistance to both gentamicin and streptomycin, tobramycin will not be effective. There are some strains that will still be amikacin or netilmicin susceptible, however, and testing for high-level resistance in these agents may be indicated. Three routine methods for the detection of HLAR: o agar dilution  500 μg/mL gentamicin and 2,000 μg/mL streptomycin  BHI agar, ambient incubation, 35°C ± 2°C, 24 hrs o broth microdilution  500 μg/mL gentamicin and 1,000 μg/mL streptomycin  BHI broth, ambient incubation, 35°C ± 2°C, 24 – 48 hrs CLSI recommends re-incubation if testing is ‘S’ after 24 hrs this is not done with agar dilution or disc diffusion  commercial automated systems are considered reliable  testing is included in most Gram positive AST panels o disk diffusion.  standard gentamicin and streptomycin disks (10mcg each) used for routine disk diffusion testing cannot be used for HLAR detection.  120 μg gentamicin and 300 μg streptomycin  MHA, ambient incubation, 35°C ± 2°C, 16 -18 hrs Fall 2024 21