Microbiology

1. MAIN ANTIMICROBIAL RESISTANCE
PATTERN IN BACTERIA: PHENOTYPES
AND GENOTYPES
: STANDARD DETECTION TECHNIQUES
AND REMEDIES
DR AVINANDA BISWAS
1ST YEAR PGT
DEPARTMENT OF MICROBIOLOGY
MALDA MEDICAL COLLEGE AND HOSPITAL

2. ANTIMICROBIAL RESISTANCE AND ITS TYPES
• Antimicrobial resistance refers to development of resistance to an antimicrobial
agent by a microorganism.
• It can be of two types : acquired and intrinsic.

3. INTRINSIC RESISTANCE
• It refers to the innate ability of a bacterium to resist a class of antimicrobial
agents due to its inherent structural or functional characteristics.
• This imposes negligible threat as it is a defined pattern of resistance and is non-
transferable .

4. EXAMPLES OF INTRINSIC RESISTANCE TO
ANTIBACTERIAL AGENTS
Natural resistance Mechanism
Anaerobic bacteria versus aminoglycosides Lack of oxidative metabolism to drive uptake
of aminoglycosides
Gram-positive bacteria versus aztreonam Lack of penicillin-binding protein (PBP)
targets that bind this beta-lactam antibiotic.
Pseudomonas aeruginosa versus
sulfonamides,trimethoprim,tetracycline, or
chloramphenicol
Lack of uptake resulting in ineffective
intracellular concentrations of these
antimicrobials.
Klebsiella spp. Versus ampicillin (a beta-
lactam) targets
Production of enzymes (beta lactamases) that
destroy ampicillin before it reaches its PBP
target.
Aerobic bacteria versus metronidazole Inability to anaerobically reduce drug to its
active form.

5. ACQUIRED RESISTANCE
• Antimicrobial resistance resulting from altered cellular physiology and structure
caused by changes in a microorganism’s genetic makeup is acquired resistance.
• Acquired resistance may be a trait associated with specific strains of a particular
organism group or species.
• This type of resistance in unpredictable.
• This unpredictability is the primary reason laboratory methods are necessary to
detect resistance patterns (also known as antimicrobial susceptibility profiles)
in clinical isolates.

6. TYPES OF ACQUIRED RESISTANCE
1.Mono drug resistance :
Resistance to one drug at a time.
Low degree resistance.
Resistance can be overcome by combination of drugs.
Virulence of resistance mutants may be lowered.
Resistance is not transferable to other organisms.
Spread to off-springs by vertical spread only.

7. TYPES OF ACQUIRED RESISTANCE (CONTD)
II. Multi drug resistance:
 Multi drug resistance at the same time.
 High degree resistance.
 cannot be overcome by drug combination.
 Virulence not decreased.
 Resistance is transferable to other organisms.
 Spread by : horizontal spread (conjugation,or rarely by transduction/transformation).
 The resistance coded plasmid (called R plasmid) can carry multiple genes,each coding for
resistance to one class of antibiotics.

8. MECHANISM OF RESISTANCE
• The following antimicrobial resistance patterns are shown in mainly gram positive
cocci:
ß lactamases enzyme
MRSA
VRSA and VISA
VRE

9. BETA LACTAMASES ENZYMES CLASSIFICATION
They can be produced by both gram-positive and gram-negative organisms.
Amber’s classification (structural or molecular classified into four classes) :
1. Class A : Extended spectrum ß lactamases (ESBL) : discuss it later
2. Class B : Metallobetalactamases : these organisms are resistant to all those
antibiotics to which AmpC beta-lactamases producers are resistant. In addition
,they are resistant to carbapenems.
3. Class C : AmpC beta lactamase : discuss it later.
4. Class D : Oxacillinase : resistance can be overcome by ß lactam + ß lactamase
inhibitor combination

10. PRODUCTION OF ß LACTAMASE ENZYME-
RESISTANCE TO ß-LACTAM ANTIBIOTICS
• Beta-lactamases genes (bla) were initially discovered on chromosomes.
• Subsequently ,they were found on plasmids .
• bla(shv) and bla(tem) genes are often located on plasmids.
• Transferred from one bacterium to other mostly by conjugation .
• Can be transferred between S.aureus strains by transduction,
• ß-lactamases or penicillinase enzymes cleave the ß-lactam rings, and thereby
organisms producing these enzymes develop resistance to ß-lactam antibiotics.

11. TREATMENT
• In this type of resistance Staphylococcus aureus is resistant to penicillin G,
Cloxacillin, Dicloxacillin.
• Addition of ß-lactamase inhibitors such as clavulanic acid or sulbactam.

12. METHICILLIN-RESISTANCE STAPHYLOCOCCUS
AUREUS (MRSA)
• Resistance to methicillin is determined by the mecA gene ,which encodes the
low-affinity penicillin-binding-protein PBP 2A.
• The mecA gene is a part of a 21-60kb staphylococcal chromosome cassette mec
(SCCmec).
• Two hypotheses have been raised to explain the evolutionary origin of
methicillin-resistant S.aureus strains.

13. METHICILLIN-RESISTANCE STAPHYLOCOCCUS
AUREUS (MRSA) (CONTD)
• The single clone hypothesis, suggests that mecA entered the S.aureus population
on one occasion and resulted in the formation of a single MRSA clone that has
since spread around the world.
• The second hypothesis, proposes that MRSA strains evolved a number of times by
means of the horizontal transfer of mecA into phylogenetically distinct
methicillin-susceptible S. aureus(MSSA) precursor strains.

14. METHICILLIN-RESISTANCE STAPHYLOCOCCUS
AUREUS (MRSA) (CONTD)
• PBP is an essential protein needed for cell wall synthesis pf bacteria. ß-lactam
drugs bind and inhibit this protein, thereby inhibiting the cell wall synthesis.
• The altered PBP2a of MRSA strains has less affinity for ß-lactam antibiotics; hence,
MRSA strains are resistant to all ß-lactam antibiotics i.e. methicillin ,oxacillin.

15. TYPES OF MRSA
Community-associated MRSA (CA-MRSA) Hospital –associated MRSA (HA-MRSA)
Express mecA gene subtype IV,V,VI Express mecA gene subtype I,II,III
More virulent and express several toxins such as
Panton Valentine (PV) toxin
They are multidrug resistant (but their virulence
is low)
They cause invasive skin and soft tissue
infections such as necrotizing fasciitis.
They cause perioperative wound infections in
hospitals and nosocomial outbreaks (hospital
staff are the major carries)

16. DETECTION OF MRSA
Test Detecting mecA-mediated resistance
cefoxitin
Detecting mec-A mediated resistance using
Test method Disc diffusion Broth microdilution Broth microdilution and agar dilution
Medium MHA CAMHB CAMHB with2% NaCl (broth microdilution)
MHA with 2% NaCl (agar dilution)
Antimicrobial
concentration
30µg cefoxitin disk 4µg/ml cefoxitin 2µg/ml of oxacillin
Inoculum Standard disc
diffusion procedure
Standard disc
diffusion procedure
Standard broth microdilution procedure or standard
agar dilution procedure
Incubation
condition
33-35⁰C ; ambient air
Incubation
length
16-18 hours 16-20 hours 24 hours (may be reported after 18 hours, if resistant
)

17. DETECTION OF MRSA (CONTD)
Test Detecting mecA-mediated resistance
cefoxitin
Detecting mec-A mediated resistance
using oxacillin
Results ≤ 21 mm= positive for
mecA mediated
resistance
≥22 mm = negative for
mecA-mediated
resistance
≥8 µg/mL=
positive for mecA
mediated
resistance
≤ 4 µg/mL =
negative for
mecA-mediated
resistance
≥ 4 µg/mL = positive for mecA-
mediated resistance
≤ 2 µg/mL= positive for mecA mediated
resistance

18. TREATMENT
• Vancomycin is the drug of choice for MRSA.
• Alternative drugs include-teicoplanin, linezolid, daptomycin, telavancin and
quinopristine/dalfopristin.
• Tetracycine ,erythromycin or cotrimoxazole may also be effective in nonlife-
threatening infections.
All ß-lactam drugs should be avoided. However,5th generation cephalosporins
,such as ceftobiprole ,ceftaroline and ceftolozane have shown some activity
against MRSA.

19. RESISTANCE TO VANCOMYCIN (VRSA AND VISA)
• Erroneous and overuse of vancomycin has led to the emergence of resistance to
vancomycin .
• It may be of low grade resistance , known as VISA (vancomycin intermediate
S.aureus) or high grade resistance ,known as VRSA (vancomycin resistant S.aureus).
• VRSA is mediated by van A gene; whereas VISA is due to increase in cell wall
thickness of S.aureus.
• The van A gene is believed to be acquired from a vancomycin-resistant strain of
Enterococcus faecalis by horizontal conjugal transfer .

20. RESISTANCE TO VANCOMYCIN (VRSA AND
VISA)(CONTD)
• Fitness cost : Acquisition of a van gene is often associated with compensatory
mutation in the gene responsible for survival which results in a reduced fitness of
S.aureus.
• In contrast, ‘fitness cost phenomena’ is not commonly observed in MRSA.
• Treatment : linezolid ,telavancin, daptomycin and quinupristin/dalfopristin are
effective drugs.

21. • The following antimicrobial resistance patterns are shown in mainly gram
negative bacilli:
ESBL
Amp C

22. VANCOMYCIN RESISTANT ENTEROCOCCI (VRE)
• Mechanism: VRE is mediated by van gene, which alters the target site for vancomycin
present in the cell wall; i.e. D-alanyl-D-alanine side chain of peptidoglycan layer
(which is the usual target site for vancomycin), is altered to D-alanyl-D-serine or D-
alanyl-D-lactate.
• This altered side chains have less affinity for binding to vancomycin.
• Van gene has several genotypes; out of which van A and van B are common types;
expressed by E. faecalis and more commonly by E. faecium.
• In India, the VRE rate reported was 9.7% (overall); 2.5% for E. faecalis and 17.4% for E.
faecium (ICMR, 2019)

23. MANAGEMENT
• As it is more common in ICU and Transplantation units infection control and
isolation precaution are needed.
• Treatment is not recommended for VRE carriers.

24. ESBL (EXTENDED SPECTRUM ß LACTAMASES)
• Mutation in the TEM and SHV genes resulting in replacement of one to six amino
acids gave more flexibility and accommodation in enzyme’s active site.
• A mutant of TEM type beta-lactamase (TEM-3/CTX-1) was detected following an
outbreak involving several K. pneumoniae isolates in Clermont-Ferrand region of
France during 1985-87.
• TEM-3 enzyme had two amino-acid substitutions (Gly→Lys at position 103 and
Gly→Ser at position 236) when compared with the parent enzyme. This enzyme
also conferred resistance to amino-, carboxy-, ureidopenicillins, aztreonam and
extended spectrum cephalosporins.

25. ESBL (EXTENDED SPECTRUM ß LACTAMASES)
(CONTD)
• SHV-2 and TEM-3 beta-lactamases were termed as extended spectrum beta-
lactamases (ESBL).
• Most ESBLs are mutants of TEM-1, TEM-2, and SHV-1, with 1- to 6-amino-acid
sequence substitutions.
• ESBLs have been observed in several members of Enterobacteriaceae (Enterobacter
spp, Salmonella spp, Proteus spp, Citrobacter spp, Morganella morganii, Serratia
marcescens, Shigella dysenteriae) as well as in Pseudomonas aeruginosa and
Acinetobacter baumannii.

26. AMP C
• Presence of a strong promoter near (upstream) the beta-lactamase gene can
overexpress the beta-lactamase production; this is often seen with CTX-M ESBL
producers.
• De-repressed strains often hyperproduce the enzyme; this is often observed
among isolates producing AmpC type beta-lactamases.
• These organisms are resistant to all those antibiotics to which ESBL producers are
resistant plus they are resistant to cephamycin (e.g. cefoxitin and cefotetan).
• But they are sensitive to carbapenems.
• Resistance cannot be overcome by ß lactam + ß lactamases inhibitor
combination.

27. CARBAPENEMASES
• Many of the OXA carbapenemase genes have been identified in environmental
isolates of P. aeruginosa, A. baumannii and Shewanella spp.
• In A. baumannii, the insertion sequences of ISAba1 type carrying strong promoters are
present upstream of chromosomal OXA genes, resulting in increased carbapenemase
expression.
• In these bacteria, OXA genes appear to be natural component of their respective
chromosomes.
• Over times, these genes would have escaped from the chromosome into mobile
genetic elements (integron, plasmids, transposons).

28. CARBAPENEMASES (CONTD)
• Mechanisms of resistance to carbapenems include production of β-lactamases,
efflux pumps, and mutations that alter the expression and/or function of porins
and PBPs.
• Combinations of these mechanisms can cause high levels of resistance to
carbapenems in bacteria such as K. pneumoniae, P. aeruginosa, and A. baumannii.
• Carbapenem resistance in Gram-positive cocci is typically due to the result of
substitutions in amino acid sequences of PBPs or acquisition/ production of a
new carbapenem-resistant PBP.

29. WHAT IS EFFLUX PUMP ?
• Certain bacteria posses efflux pumps which mediate expulsion of the drug(s) from
the cell, soon after their entry; thereby preventing the intracellular accumulation
of drugs.
• This strategy has been observed in :
E.coli and other Enterobacteriaceae against tetracyclines, chloramphenicol.
Staphylococci against macrolides and streptogramins.
S.aureus and streptococcus pneumoniae against fluroquinolones.

30. DETECTION OF BETA LACTAMASES
Antibiotic Zone diameters when testing
K. pneumoniae, K. oxytoca, E. coli P. mirabilis
Cefpodoxime 10 µg ≤ 17 mm ≤ 22 mm
Ceftazidime 30 µg ≤ 22 mm ≤ 22 mm
Cefotaxime 30 µg ≤ 27 mm ≤ 27 mm
Ceftriaxone 30 µg ≤ 25 mm -
Aztreonam 30 µg ≤ 27 mm -
:
• Screening by disk diffusion method.
• The screening test must be performed on Mueller Hinton agar on standardized
inoculum (0.5 McFarland turbidity) and diameter of the zone of inhibition must
be carefully measured after 16-18 hours of incubation at 35±2⁰C.

31. DETECTION OF BETA LACTAMASES (CONTD)
• Screening by microboth dilution method:
 Broth dilution tests must be performed on cation adjusted Mueller Hinton broth after
standardizing the inoculum.
 MIC must be observed after 16-18 hours of incubation at 35±2⁰C.
 For E. coli, K. pneumoniae, and K. oxytoca, MIC ≥ 8 µg/ml for cefpodoxime or MIC ≥ 2
µg/ml for ceftazidime, aztreonam, cefotaxime, or ceftriaxone is suggestive of ESBL
production.
 For P. mirabilis, MIC ≥ 2 µg/ml for cefpodoxime, ceftazidime, or cefotaxime is
suggestive of ESBL production.

32. DETECTION OF BETA LACTAMASES (CONTD)
Disc diffusion breakpoints MIC breakpoints
Cefpodoxime zone 9–16 mm
Ceftazidime zone 10–18 mm
Aztreonam zone 9–17 mm
Cefotaxime zone 17–25 mm
Ceftriaxone zone 16–24 mm
Cefpodoxime MIC ≥ 8 µg/ml
Ceftazidime MIC ≥ 2 µg/ml
Aztreonam MIC ≥ 2 µg/ml
Cefotaxime MIC ≥ 2 µg/ml
Ceftriaxone MIC ≥ 2 µg/ml

33. CONFIRMATORY TEST OF ESBL
1.Double Disk Synergy Test (DDST):
An amoxyclav (amoxicillin/clavulanic acid 30/10 µg) is placed at the center of the
plate. Disks containing 30 µg ceftazidime, cefotaxime, ceftriaxone, aztreonam or
10 µg cefpodoxime are placed 20-30 mm away from the central disk.
Optimal distance of disk spacing is critical for successful result. An extension in
the zone of inhibition around the peripheral disks towards the centrally placed
amoxyclav disk indicates ESBL production

34. CONFIRMATORY TEST OF ESBL (CONTD)
II. CLSI Phenotypic Confirmatory Test (PCT):
 The method recommended by CLSI can be performed as a disk diffusion or microbroth
dilution.
 In disk diffusion method ,a standardized inoculum of the test isolate is swabbed on the
surface of a Mueller Hinton agar.
 Ceftazidime (30 µg), ceftazidime/clavulanic acid (30/10 µg), cefotaxime (30 µg),
cefotaxime/clavulanic acid (30/10µg) disks are placed on the plate and incubated in
ambient air for 16-18 hours of incubation at 35±2 ⁰C.
 Confirmatory testing requires use of both cefotaxime and ceftazidime, alone and in
combination with clavulanic acid.
 An increase in the zone diameter by ≥5 mm around the disks with clavulanic acid over
the disks with cephalosporins alone confirms ESBL production.

35. III. ESBL E-test:
The Etest ESBL confirmatory test strips (AB Biodisk, Solna, Sweden) are based on the
CLSI dilution method.
The strips are thin, inert and non- porous plastic carriers, which measures 5 x 60 mm.
One side of the strip is calibrated with MIC reading scales in µg/ml while the reverse
surface carries two predefined antibiotic gradients.
There are two strips; one strip (CT/CTL) contains cefotaxime gradient at one end and
cefotaxime/clavulanic acid gradient at the other, and the other strip (TZ/TZL) contains
ceftazidime gradient at one end and ceftazidime/clavulanic acid at the other end.

36. Testing must be performed with both the strips. The MIC is interpreted as the
point of intersection of the inhibition ellipse with the Etest strip edge.
Following concentration gradients are used with these strips:
• Cefotaxime (0.25 to 16 µg/ml) AND cefotaxime/clavulanic acid (0.016 to 1 µg/ml)
plus 4 µg/ml of clavulanic acid
• Ceftazidime (0.5 to 32 µg/ml) AND ceftazidime/clavulanic acid (0.064 to 4 µg/ml)
plus 4 µg/ml clavulanic acid

37. IV.Automated methods:
• Automated ESBL tests such as Vitek Legacy and Vitek 2 (BioMérieux), MicroScan
(Siemens Medical Solutions Diagnostics), Sensititre (TREK Diagnostic Systems), and
Phoenix (BD Diagnostic Systems) offer the potential for rapid detection of ESBLs in
clinical isolates.
• The Vitek 2 and Phoenix confirmatory tests have shown be both sensitive and specific.
• Automated microdilution-based tests, such as MicroScan and Sensititre, have also
displayed the same level of accuracy as the CLSI microdilution confirmatory tests.
• These automated systems are programmed to screen as well as confirm the presence
of ESBLs.

38. DETECTION OF CARBAPENEMASES
Carbapenem Disc diffusion breakpoints (zone diameters in MIC breakpoints (µg/ml)
R I S R I S
Imipenem10µg ≥23 20-22 ≤19 ≤1 2 ≥4
Meropenem 10µg ≥23 20-22 ≤19 ≤1 2 ≥4
Ertapenem 10µg ≥23 20-22 ≤19 ≤0.25 0.5 ≥1
Doripenem 10µg ≥23 20-22 ≤19 ≤1 2 ≥4
The susceptibility breakpoints recommended by CLSI (2011;
M100-S21)