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![Laboratory detection of VRSA
• VRSA are detected by:
- Reference broth microdilution,
- agar dilution,
- E-test®,
- MicroScan® overnight and Synergies plus™;
- BD Phoenix™ system,
- Vitek 2™ system, disk diffusion, and
- The vancomycin screen agar plate [brain heart
infusion (BHI) agar containing 6 µg/ml of
vancomycin].](https://image.slidesharecdn.com/laboratorydetectionofresistantbacteria-150331053917-conversion-gate01/75/Laboratory-detection-of-resistant-bacteria-42-2048.jpg)
Uploaded byMostafa Mahmoud
Laboratory detection of resistant bacteria
The document discusses the history and mechanisms of antimicrobial resistance, including the evolution of bacterial resistance since the introduction of antimicrobials in the early 1900s. It outlines various types of resistance, including natural and acquired resistance, and specifics on detecting multidrug-resistant organisms, especially extended-spectrum beta-lactamases (ESBLs) and carbapenemases. Furthermore, it describes laboratory techniques for identifying resistant bacteria like MRSA and includes guidelines from CLSI for confirming resistance.
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Overview of the presentation topic regarding the laboratory detection of resistant bacteria by Dr. Mostafa Mahmoud.
Explains the evolution of antimicrobials from toxic metals to modern drugs, highlighting early resistance, and acknowledging the long history of resistance.
Examines the negative impact of antimicrobial resistance, including increased morbidity, mortality, treatment costs, hospital stays, and delays in interventions.
Introduces cellular targets for antimicrobials and differentiates between natural and acquired resistance mechanisms in bacteria.
Overview of different gene transfer mechanisms (transformation, transduction, conjugation) that contribute to antimicrobial resistance.
Discusses various molecular mechanisms of resistance found in bacteria, particularly focusing on Gram-negative bacteria.
Defines MDROs, mentioning common types such as MRSA, VRE, and ESBL producing Enterobacteriaceae.
Details the definition of ESBLs, detection methods, reasons for clinical concern, and CLSI testing recommendations for clinical settings.
Explains carbapenem antibiotics, causes of resistance, classification of carbapenemases, and laboratory identification approaches.
Describes MRSA identification methods, mecA gene testing, and CLSI screening tests for β-lactamase and oxacillin resistance.
Discusses the laboratory detection of VISA/VRSA, their resistance mechanisms, and the importance of reporting to infection control.
Thanks the audience and concludes the detailed discussion on resistant bacteria detection.
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Laboratory detection of resistant bacteria
- 1. LABORATORY DETECTION OF RESISTANTBACTERIA Dr Mostafa Mahmoud, MD, PHD Consultant Microbiologist Associate Prof of Microbiology & Immunology
- 2. History of antimicrobials& resistance • Bacteria are said to have been present 3,500 million years ago, causing severe morbidity and high rates of mortality before the discovery of antimicrobial agents. Antimicrobial agents were known and used for the control of bacterial infections in the form of mercury, bismuth and other heavy metals in the early 1400s, when, for example, mercury was used in the treatment of syphilis caused by Treponema pallidum. • However, These agents were toxic to the host.
- 3. • In 1908,Paul Ehrlich and colleagues discovered Salvarsan, which is an arsenic compound used in the treatment of syphilis. Ehrlich was offered the Nobel Prize for this “magic bullet”, as he termed it (Harmful Side effects). • The first conventional antimicrobial drug discovered was sulfonamide by Bayer in 1932. • This was followed in the early 1940s by the use of penicillin compounds by Florey and his colleagues in Oxford, based upon the discovery of Alexander Fleming’s natural penicillin. • streptomycin in 1944, chloramphenicol in 1947 and chlortetracycline in 1948
- 4. • In contrastto the common belief which considers antimicrobial resistance to be a new phenomenon, it is actually older. • in 1907 during the work by Ehrlich in trying to discover an agent for the drug-resistant Trypanosoma brucei, which was known as “drug- fast”. • Bacterial antimicrobial drug resistance was increasing and becoming more complicated with the introduction of further antimicrobial drugs in the 1950s, and was developed for many antimicrobials at the same time by single microorganisms which are known as multidrug- resistant (MDR) bacteria.
- 5. Effects of antimicrobialresistance: 1- increase in the rate of the morbidity and mortality related to microbial infections. 2- increase in expenses for treatment and research. 3- increase in the number of hospital stays. 4- delay in some operative interventions. 5- more costs due to isolation procedures.
- 6. Cellular targets forsome antimicrobials
- 7. Mechanisms of antimicrobialdrug resistance: A- Natural resistance • An organism is termed as having natural resistance when it is resistant to the action of an antibiotic from the start; this pattern of resistance is common to all isolates of the species, e.g. the resistance of Escherichia coli to macrolides. • It is explained by the absence or inaccessibility of the target for the drug action, e.g. Amphotericin B does not act upon bacteria due to the absence of sterols and Gram-negative bacteria are naturally resistant due to the non-permeability of the outer membrane.
- 8. • Acquired resistance •It is developed to an antibiotic to which the microorganism was previously susceptible; it develops with one or more isolates of the species, i.e. not all strains of a species are resistant. • The genetic mechanisms of acquired resistance are either due to arise of a mutation or transfer of resistant genes by conjugation, transduction, transformation or transposition.
- 9. Different gene transfermechanisms in bacteria: A =transformation, B = transduction, and C= conjugation
- 10. The molecular mechanismsof resistance:
- 11. SN Mechanism Examplesof affected antimicrobials 1 Destruction, modification, or inactivation of the antimicrobial. β-lactam antibiotics Chloramphenicol Aminoglycosides 2 Multidrug efflux pumps. Tetracycline 3 Target site alteration. β-lactam antibiotics Chloramphenicol streptomycin Quinolones Fusidic acid Erythromycin Glycopeptides Rifampicin 4 Reduction in the cell surface permeability or access of the antimicrobial to the cell interior. Tetracyclines Quinolones β-lactam antibiotics Aminoglycosides Chloraphenicol 5 New metabolic bypass mechanism. Rrimethoprim Sulphonamides
- 12. mechanisms of antimicrobialresistance in Gram-negative bacteria
- 13. MDRO: Definition • Multidrug-ResistantOrganisms (MDROs) are defined as microorganisms that are resistant to one or more classes of antimicrobial agents. • Three most common MDROs are: 1. Methicillin-Resistant Staph aureus (MRSA) 2. Vancomycin Resistant Enterococci: (VRE) 3. Extended Spectrum Beta-Lactamase producing Enterobacteriaceae. (ESBLs)
- 14. 1- Detection ofExtended-Spectrum β-Lactamases (ESBLs) production. • What are the ESBLs? • ESBL is an enzyme (constitutive, acts just when expressed) produced by certain enterobacteria (E. coli, Klebsiella & Proteus) enabling them to resist (hydrolyze) all penicillins, aztreonam, cephalosporins (including cefipime) but not cephamycins like (cefoxitin, Cefotetan) or Carbapenems.
- 15. Why should clinicallaboratory personnel be concerned about detecting these enzymes? • Difficult to detect. • Various levels of resistance to Beta Lactams.
- 16. CLSI 2010 ESBLstesting for Klebs. pneumoniae, K. oxytoca, or E. coli Disc diffusion Broth dilution Antimicrobial Content Resistant Zone Resistant MIC Initialtesting Cefpodoxime 10 μg ≤ 17 mm ≥ 8 μg/mL Ceftazidime 30 μg ≤ 22 mm ≥ 2 μg/mL Aztreonam 30 μg ≤ 27 mm ≥ 2 μg/mL Cefotaxime 30 μg ≤ 27 mm ≥ 2 μg/mL Ceftriaxone 30 μg ≤ 25 mm ≥ 2 μg/mL ConfirmatoryTesting Ceftazidime Ceftazidime - clavulanic acid 30 μg 30/10 μg A ≥ 5-mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid vs its zone when tested alone = ESBL (e.g., ceftazidime zone = 16; ceftazidime/clavulanic acid zone = 21). A ≥ 3 twofold concentration decrease in an MIC for either antimicrobial agent tested in combination with clavulanic acid (4 μg) vs its MIC when tested alone = ESBL (e.g., ceftazidime MIC = 8 μg/mL; ceftazidime-clavulanic acid MIC = 1 μg/mL). Cefotaxime Cefotaxime - clavulanic acid 30 μg 30/10 μg
- 17. CLSI recommendations forESBLs • CLSI recommends performing phenotypic confirmation of potential ESBL-producing isolates of K. pneumoniae, K. oxytoca, or E. coli by testing both cefotaxime and ceftazidime, alone and in combination with clavulanic acid. Testing can be performed by the broth microdilution method or by disk diffusion. • - For MIC testing, a decrease of > 3 doubling dilutions in an MIC for either cefotaxime or ceftazidime tested in combination with 4 µg/ml clavulanic acid added to all dilutions, versus its MIC when tested alone, confirms an ESBL-producing organism e.g. MIC to CAZ = 8; to CAZ/CLAV = 1 (8,4,2,1).
- 18. • - Fordisk diffusion testing, a > 5 mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid (10 µg) versus its zone when tested alone confirms an ESBL-producing organism. • Note there is different figures for Proteus mirabilis (see original text).
- 19. • For confirmedESßL-producing bacteria, report should express all the following as resistant even if they are susceptible in vitro: • – All penicillin’s • – All cephalosporins • - Aztreonam • For confirmed ESßL-producing bacteria, report should not override (no change in susceptibility interpretations) for: • – Cephamycins (e.g. cefoxitin). • - Beta-lactam/beta-lactamase inhibitor combinations (e.g. piperacillin/ tazobactam) • – Carbapenems
- 20.
- 21. Detection of ESBLby E-test
- 22. 2- Detection ofresistance to Carbapenems • Carbapenems are Bactericidal, beta lactam family of antibiotics, target cell wall, disrupt different stages in peptidoglycan synthesis. • The FDA approved for clinical use: imipenem, meropenem, ertapenem, and doripenem • They are broad-spectrum. • Used in treatment of life-threatening infections: - Septicemia - MDR GNB
- 23. Causes of CarbapenemResistance: 1- Impaired permeability due to porin mutation. 2- Efflux pumps. 3- “Carbapenemase-hydrolyzing enzymes” • There are 4 classes of Carbapenemases; A, B, C, and D. • Class C is very rare clinically.
- 24. Cabapenemase Ambler classificationsystem Class Enzyme Most common bacteria Inhibitor Class A Chromosomal: IMI, SME, NMC Plasmid: KPC, GES Enterobacteriaceae Boronic Acid (clavulanic) Class B Metallo-β-lactamases: IMP, GIM, VIM, SPM, NDM Ps. aeruginosa, Enterobacteriaceae Acinetobacter EDTA Class D OXA β-lactamases: Oxa-23, Oxa-48, Ps. Aeruginosa, Enterobacteriaceae Acinetobacter Oxacillin
- 25. Laboratory Approach to CarbapenemasesIdentification 1. Disc diffusion with meropenem 10 μg on Mueller Hinton agar (MHA) or broth dilution. 2. Phenotypic confirmation – Modified Hodge test – Microbiological tests with inhibitors 3. Genotypic confirmation – Molecular tests for detection of the related gene e.g. PCR.
- 26. Clinical Breakpoints forCarbapenemases detection (CLSI 2010 recommendations): Antibiotic MIC 2010 MIC Before 2010 Disc diffusion (10 μg) S I R S R S I R Imipenem < 1 2 > 4 < 4 > 16 > 23 20-22 < 19 Meropenem < 1 2 > 4 < 4 > 16 > 23 20-22 < 19 Ertapenem < 0.25 0.5 > 2 < 2 > 8 > 22 20-22 < 18 Doripenem < 1 2 > 4 < 4 ND > 23 20-22 < 19
- 27. Phenotypic confirmation Modified HodgePlate • The organism in the background of the plate is carbapenem susceptible strain (E.coli ATCC 25922) on MHA. • Drawbacks: Not identify the class – false negative
- 28. Hodge test formany strains:
- 29. Usage of carbapenemaseinhibitors: 1-Boronic Acid Synergy (combination) • Boronic acid inhibits group A Carbapenemases.
- 30. • The sametest format can be done with combination discs of EDTA, Oxacillin, for other classes of carbapenemases. • Commercial kits are available for class differentiation (Rosco kit). • Proteus spp., Providencia spp., and Morganella spp. may have elevated MICs to imipenem by mechanisms other than production of carbapenemases; thus, the usefulness of the imipenem MIC screen test for the detection of carbapenemases in these three genera is not established.
- 31. Other laboratory tests: 1–Confirmation of Meropenem resistance by E-test, 2– Colistin, Tigecycline E-tests • – No CLSI interpretative criteria 3. Genotypic confirmation (Gold Standard) • PCR assays can target different genes in single or multiplex formats. – Class A (KPC, IMI, NMC, SME), – Class B (IMP, VIM, AIM, GIM, KHM, SIM, SPM, and NDM) – Class D (OXA-23-like, OXA-24-like, OXA-48-like, OXA-51-like and OXA-58-like)
- 32. 3- MRSA Screening •The MRSA are either healthcare associated (HA- MRSA) or community acquired (CA-MRSA). • It is resistant to anti-beta lactamase penicillin e.g. oxicillin, methicillin. Cloxacillin, flucloxacillin, • The mechanism of resistance is via chromosomally mediated mecA gene which mediates the expression of a different penicillin Binding protein called (PBP-2a) which does not bind these drugs making the organism resist to them.
- 33. • MRSA screeningdepend upon PCI policy. • Criteria for MRSA: 1- The presence of the mecA gene. 2- Either an oxacillin MIC of > 2 μg/mL, a methicillin MIC of > 4 μg/mL, or a cefoxitin MIC of > 4 μg/mL. (The most reliable one is cefoxitin).
- 34. Detection methods are: 1-Conventional by culture on: – Mannitol slat agar (MSA) with 7% NaCl or chromogenic agar with high salt content. – Enrichment on nutrient broth with high salt content then subculture on MSA. 2- Molecular method: by detection of the mecA gene by PCR. • MSA is not suitable for running serological testing for identification of Staph aureus also slow growth.
- 35.
- 36. MRSA chromogenic agar(Hardy)
- 37. Brilliance MRSA 2Agar: Rapid & Reliable MRSA Screening in Just 18 H (Oxoid)
- 38. BD chromogenic agarfor MRSA (in 20 h – red colonies are MRSA)
- 39. CLSI Screening Testsfor β-Lactamase Production, Oxacillin Resistance, and mecA- Mediated Oxacillin Resistance Using Cefoxitin in the S. aureus. Screen test β-Lactamase Oxacillin Resistance mecA-Mediated Oxacillin Resistance Using Cefoxitin Organisms S. aureus with penicillin MICs ≤ 0.12 μg/mL or zones ≥ 29 mm S. aureus S. aureus and S. lugdunensis Test method Disk diffusion Nitrocefin-based test Agar dilution Disk diffusion Broth microdilution Medium MHA NA MHA with 4%NaCl MHA CAMHB AM Conc. 10 U disc penicillin NA 6 μg/mL oxacillin 30 μg Disc of cefoxitin 4 μg/mL cefoxitin Inoculum Standard disk diffusion Induced growth1 Direct colony suspension2 Standard disk diffusion Standard broth microdilution Incubation conditions 35 ± 2°C; ambient air Room temperature 33–35°C; ambient air.(Testing at emperatures above 35°C may not detect MRSA.) Incubation length 16–18 hours Up to 1 hour 24 hours; 16–18 hours 16–20 hours Results Sharp zone edge (“cliff”) =β-lact. positive. Fuzzy zone edge (“beach”) =β-Lact. negative. Nitrocefin-based test: conversion from yellow to red/pink = β-lactamase positive. Examine carefully with transmitted light for > 1 colony or light film of growth. > 1 colony = oxacillin resistant. ≤ 21 mm = mecA positive ≥ 22 mm = mecA negative > 4 μg/mL = mecA positive ≤ 4 μg/mL = mecA negative
- 40. Screen test β-LactamaseOxacillin Resistance mecA-Mediated Oxacillin Resistance Using Cefoxitin Further testing and reporting β-Lactamase-positive staphylococci are resistant to penicillin, amino-, carboxy-, and ureidopenicillins. Oxacillin-resistant staphylococci are resistant to all β-lactam agents; other β- lactam agents should be reported as resistant or should not be reported. Cefoxitin is used as a surrogate for mecA-mediated oxacillin resistance. Isolates that test as mecA positive should be reported as oxacillin (not cefoxitin) resistant; other β-lactam agents should be reported as resistant or should not be reported. Because of the rare occurrence of oxacillin resistance mechanisms other than mecA, isolates that test as mecA negative, but for which the oxacillin MICs are resistant (MIC ≥ 4 μg/mL), should be reported as oxacillin resistant. QC recommend -ations S. aureus ATCC® 25923 S. aureus ATCC® 29213 S. aureus ATCC® 29213 positive S. aureus ATCC® 25923 negative S. aureus ATCC® 29213 – Suscep. ATCC® 43300 – Resistant S. aureus ATCC® 25923 mecA negative (zone 23–29 mm) S. aureus ATCC® 43300 mecA positive (zone ≤ 21 mm) S. aureus ATCC® 29213 mecA negative (MIC 1–4 μg/mL) S. aureus ATCC® 43300 mecA positive (MIC > 4 μg/mL)
- 41. 4- Vancomycin-Intermediate/Resistant Staphylococcus Aureus(VISA/VRSA) laboratory detection • Most SA are VSSA with MIC of 0.5-2 μg/mL. • Vancomycin MIC of VISA is 4-8 μg/mL. • Vancomycin MIC of VRSA is ≥ 16 μg/mL. • No CLSI disc diffusion criteria for VISA or VRSA (only for VSSA >=15 mm).
- 42. Laboratory detection ofVRSA • VRSA are detected by: - Reference broth microdilution, - agar dilution, - E-test®, - MicroScan® overnight and Synergies plus™; - BD Phoenix™ system, - Vitek 2™ system, disk diffusion, and - The vancomycin screen agar plate [brain heart infusion (BHI) agar containing 6 µg/ml of vancomycin].
- 43. What are themechanisms of resistance for VRSA and VISA? • All VRSA isolates to date contained the vanA vancomycin resistance gene. • The vanA gene is usually found in enterococci and typically confers high-level vancomycin resistance (MICs= 512-1024 µg/ml) to these organisms (VRE). • There is also VanB and VanC genes.
- 44. Should VISA andVRSA be reported to the infection control team? • Yesand you have to notify also the lab admin about any suspicious isolates.
- 45.