Presentation to guide the microbiology lab personnels regarding the diagnosis of multidrug-resistant bacteria MDR bacteria.

1. LABORATORY DETECTION
OF RESISTANT BACTERIA
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 contrast to 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 antimicrobial resistance:
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 for some antimicrobials

7. Mechanisms of antimicrobial drug 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 transfer mechanisms in bacteria: A
=transformation, B = transduction, and C= conjugation

10. The molecular mechanisms of resistance:

11. SN Mechanism Examples of 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 antimicrobial resistance in
Gram-negative bacteria

13. MDRO: Definition
• Multidrug-Resistant Organisms (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 of Extended-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 clinical laboratory
personnel be concerned about detecting
these enzymes?
• Difficult to detect.
• Various levels of resistance to Beta Lactams.

16. CLSI 2010 ESBLs testing 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 for ESBLs
• 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. • - For disk 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 confirmed ESßL-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. Phenotypic detection of ESBL

21. Detection of ESBL by E-test

22. 2- Detection of resistance 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 Carbapenem Resistance:
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 classification system
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
Carbapenemases Identification
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 for Carbapenemases
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 Hodge Plate
• 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 for many strains:

29. Usage of carbapenemase inhibitors:
1-Boronic Acid Synergy (combination)
• Boronic acid inhibits group A
Carbapenemases.

30. • The same test 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 screening depend 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. Mannitol Salt agar

36. MRSA chromogenic agar (Hardy)

37. Brilliance MRSA 2 Agar: Rapid & Reliable
MRSA Screening in Just 18 H (Oxoid)

38. BD chromogenic agar for MRSA
(in 20 h – red colonies are MRSA)

39. CLSI Screening Tests for 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-Lactamase Oxacillin 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 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].

43. What are the mechanisms 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 and VRSA be reported to
the infection control team?
• Yesand you have to notify also the lab
admin about any suspicious isolates.

45. Thank You