Q1 & 2: HA-MRSA & CA-MRSA

Definition:
Methicillin resistance S. aureus is defined by CLSI as a S. aureus with an oxacillin Minimum
inhibitory concentration of greater or equal to 4mcg/mL. MRSA requires the presence of mecA
gene and is subdivided into HA-MRSA ad CA-MRSA.
Four mechanisms can confer resistance towards methicillin:
- Expression of acquired chromosomal mecA gene to produce supplemental PBP (known as
PBP2a) with low affinity binding for penicillins.
- Production of PBP variant, called mecC found mainly in livestock
- Inactivation of drug by increased production of beta lactamases
- Production of modified intrinsic PBPs with altered affinity for the drug.

Epidemiology, infected individuals & clinical presentation:

HA-MRSA: CA-MRSA:
- Defintion: MRSA infection that occurs > - Definition: an MRSA infection that occurs in
48hours after hospitalization or that occurs the absence of health care exposure.
outside of hospital within 12months after
- Often associated with SSTI in young, healthy
health care exposure (ie history of surgery,
adults.
recent hospitalization etc).
- Predominantly causes skin and soft tissue
- occurs in the elderly
infection, but also causes invasive, severe
- It is associated with severe, invasive disease, disease such as necrotizing pneumonia, IE, UTI,
including bacteremia, pneumonia & SSTI. osteomyelitis and sepsis (with or without
Waterhouse-Friderichson syndrome).
- Occurs in hospital settings due to presence of
invasive device such as urinary catheter, CVC - Community outbreaks can occur. Person
etc. colonized with MRSA also can transmit disease.
Animals can carry MRSA and serves as source
- STAU has ability to form biofilm and adhere
of transmission.
to surfaces.
- CA-MRSA may also cause hospital onset
MRSA. Patients colonised from the community
& admitted due to CA-MRSA infection can
transmit it to other patients in the hospital, as
well as in health-care associated setting.
Diagnosis & AST pattern:
 Molecular mechanism of resistance:
- Methicillin resistant is mediated by PBP2a, which is encoded by mecA that is located in
mobile genetic element called the Staphylococcal chromosome cassette (SCCmec).
- PBPs are peptidase enzymes located in bacterial membrane which catalyses
transpeptidation of cell wall synthesis.
- in MSSA strains, BL binds to PBP1-3, inhibiting transpeptidation and thereby inhibiting cell
wall assembly.
- PBP2a has low affinity for BL antibiotics. PBP2a can substitute enzymatic activity of
PBPs (normal ones) and complete cell wall assembly.
 AST pattern:
a) HA-MRSA: b) CA-MRSA:

- Majority are multidrug - Mostly sensitive to non-beta lactam

resistant & caries SCCmec antibiotics, ex clindamycin, Bactrim &
type I, II, and III. ciprofloxacin.
- However, incidence of multidrug resistance
CA-MRSA has been increasing.
- CA-MRSA carries SCCmec type IV and V
as well as cytotoxic Panton-Valentine
Leukocidin which confers enhance
virulence.
 Expression of methicillin resistance: homogenous, heterogenous and borderline.
- Strains with mecA can expresses homogenous or heterogenous resistance.
- homogenous are resistance to all when tested with in vitro methods.
- heterogenous isolate results in subpopulation of cells that appear to be susceptible and others
resistance & is more common in CoNS.
- Heterogenous expression results in MIC near breakpoint (2-8ug/ml), & can be misinterpret
as susceptible.
 BORSA & MOD-SA also results in oxacillin MICs near breakpoint.
- BORSA can be distinguished from mecA or MOD-SA resistance by adding BLI (eg
clavulanic acid) to oxacillin MIC test, which lowers the MIC by 2 dilutions or more.
- BORSA or MOD-SA usually do not have multiple drug resistance.
 Diagnosis: (CLSI)
a) Phenotypic tests:
- 3 types of tests to detect mecA mediated resistance
Tests Comments Advantages Limitations
i) Cefoxitin - DD more reliable method because it is - easy to read Does not detect
disc diffusion a better inducer of mecA gene than using reflected BORSA & MOD-
or broth oxacillin. light. Performs SA, only oxacillin
microdilution well for detection detects these
- Also, a surrogate for oxacillin in
of borderline resistance
routine AST methods.
resistance cause mechanisms.
- STAU colonies are cultured on by mecA & mecC
MHA/CAMHB with standard in STAU.
procedure.
- a diameter of <=21mm
(DD)/>=8microgram/mL (broth
microdilution) indicates positive for
mecA-mediated resistance,
- >=22mm/<=4microgram/mL
indicates negative results.
ii) Oxacillin - Method: Broth microdilution & agar
broth dilution.
microdilution
- Broth microdilution: CAMHB with
& agar dilution
2% NaCl
- Agar dilution: MHA with 2% NaCl
- >=4ug/mL -> positive for mecA
mediated resistance
- <= 2ug/mL-> negative
iii) Oxacillin - MHA with 4% NaCl - less sensitive
salt agar than cefoxitin DD
- >1 colony=positive for mecA
test.
mediated resistance
- may not detetct BORSA with non-
mecA mediated resistance mechanism.
 Treatment: (based on UK & IDSA guidelines)
Disease Treatment option
1. - Vancomycin 6mg/kg/dose IV for at least 2 weeks OD
Bacteremia - if vanco contraindicated, linezolid can be administered as an alternative first
(UK) line treatment.
- if first line is contraindicated, daptomycin or teicoplanin is an alternative
treatment.
- Bactrim can be used as oral step down but not as first line treatment.
- uncomplicated: duration 14 days.
- complicated: 28 days at least.
2. Infective I, Native - vancomycin or daptomycin 6mg/kg/dose OD for 6 weeks.
endocarditis - ECHO (preferably TEE) should be performed.
(IDSA) Ii, Prosthetic - Vancomycin 6mg/kg/dose OD IV plus rifampicin 600mg PO/IV TDS 6weeks
valve & Gentamycin 1mg/kg/dose TDS 2 weeks

3. I, Necrotizing - vancomycin or linezolid

Pneumonia pneumonia - consider adding on toxin inhibiting agent, clindamycin or rifampicin if
susceptible.
Ii, Nosocomial - vancomycin or linezolid
pneumonia - not recommended to give daptomycin as it is inhibited by lung surfactant.
* Duration: 7-21days (IDSA)

4. SSTI I, - Incision and drainage are the mainstay of treatment.

Uncomplicated - no need antibiotics if no systemic illness, not immunocompromised or
abscess abscess not extensive (<5cm and already drained).

Ii, Complicated - vancomycin IV, oral or IV linezolid, IV daptomycin, IV or oral clindamycin

600mg TDS (IDSA).
* Use oral Bactrim or clindamycin when treatment is warranted and if
susceptible (UK).
- Duration: 7-14days.

IIi, Impetigo - topical antiseptic ex hydrogen peroxide 1%, if localized and non-bullous
and pt clinically well.
- to prevent resistance, mupirocin or fusidic acid can give as second line.
- complicated: to give systemic antibiotics.
Iv, Cellulitis or - Severe: vancomycin or teicoplanin as first line
skin and soft - Alternative: daptomycin or linezolid.
tissue infection - 1st and second line contraindicated: Tigecycline
(UK) - Mild: oral Bactrim, clindamycin, doxycycline can be given in mild soft tissue
infection or oral step- down therapy.

V, Recurrent - Decolonization should be considered in patients with recurrent MRSA despite

MRSA SSTI strict hygiene measures and wound care.
- Nasal decolonization includes applying mupirocin ointment BD for 5 -10days
and decolonization bath (eh chlorhexidine bath) for 5-14days or dilute bleach
 baths.

5. Bone & - surgery and drainage where indicated.

joint - IV vancomycin & teicoplanin for 2 weeks as first line therapy followed by
infection further IV or oral antibiotics to complete 4 weeks in septic arthritis or 6 weeks
in osteomyelitis.
- if glycopeptide contraindicated, linezolid or daptomycin.
- oral Bactrim, doxycycline, linezolid or clindamycin as oral stepdown if
susceptible.
- rifampicin, fusidic acid and quinolones should not be used as monotherapy but
in combination with other susceptible drugs.

6. UTI (UK) - Exclude MRSA bacteremia before initiating treatment.

- once excluded, lower UTI can be treated with oral Bactrim, doxycycline and
ciprofloxacin if susceptible.
- if complicated, it can be treated with IV vancomycin or teicoplanin.
- if contraindicated, second line is daptomycin.
- Linezolid should not be used as it has poor renal excretion.
- in CAUTI, whenever possible/ practicable, replace the catheter with or
without single dose of gentamycin (if susceptible). If gentamycin
contraindicated, single dose of IV vancomycin or teicoplanin can be given.
6. CNS I, intracranial or - Adequate source control is required. If contraindicated, incision and drainage
infection spinal infections. for treatment (eg subdural emphyema or brain abscess).
- Antibiotics alone would suffice in patients with small epidural abscess and
absence of neurological deficit.
- IV vancomycin and linezolid is first line for treatment.
- Duration of treatment: 4-6 weeks in subdural empyema, brains abscess, septic
thrombosis of cavernous sinus.

Ii, Meningitis - first line is vancomycin IV. In severe infections, consider adding on
rifampicin if susceptible.
- if not responding, consider intrathecal vancomycin into ventricles.
- duration of treatment: 2 weeks.
* Linezolid & Bactrim is an alternative treatment for CNS infection (IDSA).
* UK guidelines does not recommend use of linezolid, clindamycin and
chloramphenicol as these drugs are not bactericidal.
*** Linezolid as alternative but limited due to toxicity and certain
population/peads.
* Daptomycin not indicated due to poor penetration into CSF.
* In CNS shunt infection, shunt should be removed and replaced only when
culture is repeatedly negative.

Prevention:
Health-care settings
 Principles Comments
1. Basic infection - Hand hygiene should be strictly adhered when providing care for patient infected with
prevention MRSA. Hand hygiene consists of cleaning hands with soap and water or alcohol-based hand
principles. rub according to WHO 5 moments hand hygiene.
- Contact & standard precaution consists of wearing gowns & gloves mask during clinical
encounter with MRSA patients & body fluids.
- Patients infected or colonised with MRSA should be cohorted together/ isolated into single
rooms. If possible, medical equipment (eg stethoscope) should be dedicated to single
patient. If not possible, equipment should be cleaned and disinfected.
- Discontinuation of contact precaution is uncertain. SHEA suggests discontinuation after
documented 1 to 3 negative weekly surveillance cultures in patients infected or colonised
with MRSA who are not on antibiotics active against MRSA.
- CDC recommends discontinue contact precautions when three or more surveillance
cultures are negative over the course of a week or two in the absence of antimicrobial
therapy (for several weeks), a draining wound, respiratory secretions, or evidence
implicating the patient in ongoing transmission.

2. Active - Definition: active surveillance consists of performing screening cultures (eg nasal swab,
surveillance. oropharynx &/or perineum) to identify asymptomatic patients to minimize transmission.
- Methods: standard microbiological method, selective media, PCR. Rapid WGS is an
alternative method that may be useful for outbreak purposes but not routinely available.
- Active surveillance cultures are more useful in outbreak situations and in patients with
high risk of MRSA infection such as patients in ICU, immunocompromised, history of
admission in previous 12 months, receive antibiotic in previous 3 months, patients with
history of MRSA colonization (should be isolated while waiting for surveillance culture
results), in long term care facilities, has wounds during admission and haemodialysis.

3. Decolonization - CHG 4% bath (body washing) for 5 days and hair to wash at day 1 and 5.
- Mupirocin 2% nasal ointment BD daily for 5 days.
- Rescreen at day 7.
- Do not screen during treatment.

4. Education - HCW should be educated regarding risk factors, routes of transmission, outcomes and
infection prevention strategies.
- Patient and family members should be educated to reduce anxiety, improve satisfaction
and promote adherence to institution contact policies.

5. - Cleaning and disinfection should be done on patient’s care environment (eg bed rails,
Environmental bedside table, high touch surfaces) with special attention to adequacy of cleaning, dilution
cleaning of disinfectant and duration of contact of disinfectant with surface.
 Chlorhexidine gluconate 4% daily
skin wash and neomycin nasal
cream (Naseptin) applied to the
anterior nares four times daily for 5
days in mupirocin resistant cases.

Prevention of CA-MRSA
Preventive measures -Hand hygiene
-Other measures: wounds that are draining should be kept covered with
clean and dry bandages.
- Activities involving skin to skin contact should be avoided. Patients
should not share personnel items such as towels, razor etc.
-Decolonization (same like HA-MRSA)

**Goals of Antimicrobial Stewardship (List Down-General):

 Global Definition: AMS is any activity to optimize drug selection, dose, duration and route
of an antimicrobial.

Consists of 4 goals:
- Antimicrobial selection encompasses the narrowest spectrum of therapy to treat the
suspected organism that also penetrates the site of infection. Appropriate selection includes
empiric therapy, which takes into account likely pathogens, local susceptibility data, and
patient-specific factors, such as allergies, severity of illness, and comorbidities. Once a
causative pathogen is identified therapy should be de-escalated to the narrowest spectrum
therapy that covers the pathogen.
- Dose optimization includes strategies for optimizing likelihood of achieving pharmacokinetic
or pharmacodynamic targets (eg, extended infusion of beta-lactams) or dose reduction in the
presence of renal insufficiency to ensure that patients do not have elevated concentrations of
the antimicrobial, which can be associated with increased toxicity.
- Appropriate route includes transitioning patients to oral therapy once indicated.
- Optimal duration is based on the clinical course of the infection, in combination with
evidence-based guidelines.

Q3: heteroresistant VISA, VISA, VRSA

Definitions:

CLSI breakpoints:
EUCAST breakpoint:
Vancomycin
. susceptible: ≤2 mcg/mL
Vancomycin intermediate: 4 to 8 mcg/mL Vancomycin susceptible: ≤2 mcg/mL
Vancomycin resistant: ≥16 mcg/mL Vancomycin resistant: >2 mcg/mL

When to consider emergence of VISA/VRSA/hVISA??

- when there’s repeated isolation of STAU from normally sterile site after 3 to 4 days of treatment,
despite adequate source control.
- However, persistent bacteremia may reflect an unrecognized abscess or metastatic infection rather
than reduced antibiotic susceptibility.
 Heteroresistant VISA
- refers to VISA strains in which subpopulations display variable rather than uniform susceptibility
to vancomycin.
- Heteroresistant strains of STAU contain subpopulations of bacteria with vancomycin MICs in the
intermediate range, while the vancomycin MIC for the entire population of the strain remains within
the susceptible range.

Mechanism of resistant:
Mechanism
a) - Increase in the minimum inhibitory concentration (MIC) of methicillin-resistant MRSA isolates
Borderline to vancomycin (known as "MIC creep")
vancomycin
- not necessarily reached the level of vancomycin resistance as VISA, but they have been at the
susceptibilit
upper limits of the susceptible range.
y
b) VISA - unusually thickened cell wall containing excessive vancomycin target dipeptides (D-Ala-D-
Ala) capable of "trapping" vancomycin, thereby reducing availability of the drug for cellular
targets.
- involves the stepwise acquisition of multiple mutations in genes that are two component
regulatory systems.
- Gene involved (based on studies): vraR, graRS, and walRK
- VISA strains often develop from MRSA strains that are exposed to vancomycin for prolonged
periods of time.
c) VRSA - emerged via vanA gene plasmid-mediated transfer from enterococci with vancomycin resistance
to S. aureus via mobile genetic element Tn1546
- Resistance is due to the synthesis of an alternative cell wall terminal peptide (D-ala-D-lac) rather
than the normal terminal peptide (D-ala-D-ala)àVancomycin is unable to bind to the first peptide.

Epidemiology:
VISA
- rare
- RF: ongoing or recent dialysis, MRSA bacteremia associated with CVC or prosthetic graft material,
and prolonged vancomycin exposure (6 to 18 weeks) in the three to six months preceding infection.

VRSA
- scattered reports around the world.
- Most of the VRSA isolates were from skin and soft tissue sites, and the majority of case patients
were also colonized or infected with a VRE.
- Because cocolonization with MRSA and VRE is common, development of further VRSA strains
remains a concern.
- Although the majority of VRSA isolates reported to date have been from health care-associated
MRSA strains, a few have been associated with community-acquired strains , indicating the
possibility of transferring plasmid-mediated vancomycin resistance to various MRSA strains.
Secondary spread of these strains to health care workers appears to be limited.

Laboratory testing:
1) Detection of VRSA & VISA
VRSA (CDC)
- VRSA are detected by reference broth microdilution, agar dilution, Etest®, MicroScan® overnight
and Synergies plus™; BD Phoenix™ system, Vitek2™ system, disk diffusion, and the vancomycin
screen agar plate [brain heart infusion (BHI) agar containing 6 µg/ml of vancomycin].

VISA (CDC)
- VISA isolates are not detected by disk diffusion because these isolates produce zone diameter within
the susceptible range (>=15mm). à CLSI removed
- Methods that typically detect VISA are non-automated MIC methods including reference broth
microdilution, agar dilution, and Etest® using a 0.5 McFarland standard to prepare inoculum.
- Laboratories using disk diffusion to determine vancomycin susceptibility should consider adding a
second method for VISA detection.
- The vancomycin screen plate is useful for detecting VISA (MIC = 8 µg/ml). Reliable detection of
VISA (MIC = 4 µg/ml) may require a non-automated MIC method.

CLSI approved vancomycin screen agar

- Perform if vancomycin MIC >=8ug/mL
- Method: Agar dilution using BHI agar with 6ug/mL vancomycin concentration.
Prepare 0.5mcF suspension, drop on agar plate and inoculate.
>1 colony or light film of growth àpresumptive reduced susceptibility to vancomycin.
Perform vanco MIC on the colonies that grew on BHI- vanco screen agar.
* Testing on BHI-vanco agar does not reliably detect all VISA. Some strains to which the
vanco MIC are 4ug/mL will fail to grow.
Growth on agar plate should be checked for purity.
2) Detection of Heteroresistant VISA (manual of clinical microbiology)
- Some hVISA are detetcted by standard methods because the population MIC is 4ug/mL.
- Some continued to be missed because population MIC is <=2ug/mL.
- Gold standard for detection à population analysis/area-under-the-curve analysis (not available in
labs).
- 2 Etest have been developed:
 Macro Etest
o uses large inoculum spread on BHI agar plates & separate vanco & teicoplanin strips.
o MIC of >=8ug/mL either strip or MIC >=12ug/mL for teicoplanin is considered
POSITIVE.
o Sensitivity 57-89%
 Etest GRD
o Performed using MHA with 5% sheep blood
o Inoculum equivalent to 0.5McF, & single strip impregnated with both vanco &
teicoplanin àinterpret specific MIC àresult either positive/negative
o Sensitivity 57-100%.

Treatment (up to date)

1) Borderline vancomycin susceptiblity
- if vanco approach MIC limit of 2ug/mL & poor initial clinical response à discontinue vancomycin
& switch to daptomycin.
- use of combo therapy:
o Daptomycin plus ceftaroline
o Vancomycin plus ceftaroline or other beta-lactams
o Daptomycin plus trimethoprim-sulfamethoxazole
o Ceftaroline plus trimethoprim-sulfamethoxazole

- Alternative monotherapy: televacin, ceftaroline, linezolid

2) VISA & VRSA

- optimal regimen uncertain.

- Monotherapy is reasonable for patients with bacteremia where there is source control and proven
absence of deep-seated infection.
- Combination therapy is warranted for patients with bacteremia and concomitant deep-seated
infection to minimize the likelihood of emergence of resistance during therapy
- Possible combination regimens include (depending on susceptibility testing)
o Daptomycin plus ceftaroline or other beta-lactams
o Vancomycin plus ceftaroline or other beta-lactams
o Daptomycin plus trimethoprim-sulfamethoxazole
o Ceftaroline plus trimethoprim-sulfamethoxazole

- Possible monotherapy regimens include telavancin, ceftaroline, and linezolid

BORSA (from hafsyah’s ppt)

Definition:
- Low, borderline resistance to penicillinase-resistant penicillins (PRPs)-Oxacillin, Cloxacillin,
Dicloxacillin, Nafcillin.
- Oxacillin MICs typically equal to 2–8 µg/ml.
- In contrast to methicillin-resistant S. aureus (MRSA), BORSA do not have an altered penicillin-
binding protein, PBP2a, encoded by the mecA or mecC gene.

Epidemiology:
- The prevalence of BORSA strains is approximately 5% on average, but may vary from population to
population (1.4%–12.5%).
- However, the exact prevalence is difficult to establish and it is underestimated because many clinical
microbiology laboratories use only the cefoxitin test for detection of oxacillin resistance in
Staphylococcus spp.
- Cefoxitin test is a marker of resistance to oxacillin by acquisition of the mecA gene and it is unable
to detect BORSA strains.

Classification on basis of susceptibilities to beta-lactam antimicrobial agents

- Penicillin – Sensitive (>90% S.aureus are resistant to Penicillins)
- Penicillin – Resistant, Methicillin – Sensitive
- Methicillin - Resistant

Mechanisms of Methicillin (Oxacillin)- Resistance S.aureus

- Expression of acquired chromosomal mecA gene that encodes a low-affinity penicillin-binding
protein (PBP) designated PBP2a or PBP2’. (most common)
- Production of different PBP variant (eg mecC gene,encode PBP2c - found in livestock (LA-MRSA),
low prevalence in human <2.8%)
- Inactivation of drug by production of beta-lactamase (BORSA)
- Production of modified intrinsic PBPs with altered affinity for the drugs (MOD-SA : rare)

Mechanisms of Borderline Resistance S.aureus

- Hyperproduction of ß-lactamases – BORSA (Borderline Oxacillin Resistant S.aureus) / Borderline
methicillin susceptible –leads to hydrolysis of Penicillinase-stable penicillins.
- Inducible, plasmid-mediated methicillinase or
- Different modifications in the PBP genes due to spontaneous amino acid substitutions in the
transpeptidase domain that leads to altered binding affinity of normal penicillin-binding proteins
(PBPs) - MODSA (Modified S.aureus) / Low-level methicillin resistant.

CLINICAL SIGNIFICANCE
- BORSA have been reported to be associated with both nosocomial & community-acquired
infections.
- It has been isolated from various infections site including skin and soft tissue infections, surgical
wounds, respiratory samples, and respiratory tract.
- It also can cause more severe infections, such as endocarditis and sepsis.
- Few cases of BORSA reported were isolated in dermatology unit in a hospital setting.

Cefoxitin Vs Oxacillin
- Oxacillin disc diffusion has been the traditional method for methicillin resistance screening.
- Now, Oxacillin disc diffusion is not recommended & not reliable, because incomplete induction of
mecA results in light/hazy growth in zone of inhibition, affecting reading of zone diameter.
- This test often fails to detect heterogeneous MRSA populations.
- 30-µg cefoxitin disc test has proven to be more efficient in predicting methicillin resistance.
Cefoxitin is a more potent inducer of mecA, easier to read, therefore more sensitive/specific in
detecting MRSA in disc diffusion.

CLSI Disc diffusion and BMD methods for MRSA detection

Oxacillin DD: Not recommended

Oxacillin MIC: S</= 2, R>/=4
Cefoxitin DD: S >/=22, R </=21
Cefoxitin MIC: S</=4, R>/=8

METHODS FOR DETECTION OF BORSA

- Routine diagnostics detection method of BORSA still underdeveloped.
- Due to lack of such methods, some BORSA strains may be misdiagnosed as MRSA or MSSA
isolates.
-These strains were not detected by cefoxitin test, to detect all strains resistant to oxacillin laboratories
should routinely test for both cefoxitin & oxacillin.
- SUSPECT BORSA WHEN OXACILLIN MIC (MBD / E-TEST) ≥ 4 mcg/ml AND CEFOXITIN
SCREENING NEGATIVE

Detection of ß-lactamase production in Staphylococcus spp.

To detect hyperproduction of beta-lactamases, 2 methods that can be employed:
- Disk-diffusion (penicillin zone-edge test)
- Cefinase test with a nitrocefin disc.

1)Disk-diffusion (penicillin zone-edge test)

- It is a standard disc diffusion procedure using 10 units penicillin disk, incubated 35 ±2°C, ambient
air for 16 – 18 hours.
- It was shown to be more sensitive than nitrocefin-based tests for detection of ß-lactamase production
in Staphylococcus spp.
- Results interpretation:

2)Nitrocefin disk test Fuzzy zone edge - ß-lactamase negative

Sharp zone edge - ß-lactamase positive

Nitrocefin-based tests can be used for S. aureus, but negative results should be confirmed with the
penicillin zone-edge test before reporting penicillin as susceptible.

Oxacillin + Clavulanic acid

- BORSA strains become fully susceptible to oxacillin in the presence of beta-lactamase inhibitor.
- Significant increases (5mm) of the inhibition zone diameter for oxacillin were shown after the
addition of 4 µg of clavulanic acid, OR
- 2-fold dilution decreases in oxacillin MICs were obtained in the presence of 4 µg/ml of clavulanic
acid.
- Both of these parameters remain unchanged in MSSA and MRSA.

Molecular methods
- β-lactamase is encoded by a gene called blaZ.
- Two other genes regulate the expression of blaZ: a repressor (blaI) and an anti-repressor (blaR1).
- Due to hyperproduction of beta-lactamases by BORSA strain, blaZ gene can be detected by
molecular methods.
- In one study, blaZ derived from BORSA was found to have decreased susceptibility to oxacillin
compared with a strain carrying blaZ from an MSSA strain, with 6 differences in amino acid sequence
between MSSA and BORSA.

Oxacillin salt agar (CLSI 30th edition)

- To detect mec-A mediated resistance.
- Prepare colony suspension of 0.5McFarland, using a 1-µL loop dipped in the suspension, spot
inoculation an area 10-15mm in diameter into onto MHA plate containing 6 µg/ml OXA and 4 %
NaCl, incubate at 35 °C.
- If no growth was observed at 24 hours, plates were incubated for an additional 24 hours.
- Strains that grew on the agar were considered resistant (MRSA).
- >1 colony = Oxacillin resistant
- The incubation temperature should not exceed 35°C, as MRSA may not be detected in warmer
temperature.
- This test may not detect low frequency of resistant expression by heteroresistant mec-A positive
strains & may also not detect borderline-resistant strains with non-mecA mediated resistance
mechanisms

TREATMENT
- The treatment of choice is large doses of penicillinase-resistant penicillins (e.g Cloxacillin) OR beta
lactams + beta lactamase inhibitors (e.g ampicillin/sulbactam).
- However, it poses challenges when if PRPs even at higher doses are inefficient in the treatment of
severe infections.
- BORSA should also be suspected if infections do not respond to PRPs.
- If unable to differentiate between these two phenotypes, each infection should be treated as MRSA.

Q4: ESBL
Definition:
Extended spectrum beta lactamases are enzymes that confer resistance to most BL
antibiotics including penicillin, cephalosporins and monobactam aztreonam.
- Varies in activity against different oxymino-beta-lactam substrates but cannot attack
cephamycins (ex: cefoxitin) and cabapenems.
- Generally susceptible to beta-lactam inhibitors, such as clavulanic acid, sulbactam,
tazobactam.
- Found exclusively in GN organisms, mainly KLPN, K.oxytoca & E.coli but also in
Acinetobacter, Burkholderia, Citrobacter, Enterobacter, Morganella, Proteus, Pseudomonas,
Salmonella, Shigella, Serratia.

- ESBL varieties:
Varieties Description
TEM beta- - Occurs due to amino acid substitution around active site of enzyme
lactamases: that change its configuration.
- Not all will behave like ESBL, ex TEM1 & 2, only hydrolyses BL
such as penicillins & narrow spectrum cephalosporins.
- However, most are ESBLs, some are resistant to BL inhibitors &
few are both ESBL & inhibitor resistant.

SHV BL: - Due to amino acid changes around site. Found worldwide.
- Most common SHV 2, 5, 7 & 12.
 - Not all are ESBL, some for ex SHV 1 only hydrolyses BL such as
penicillin & narrow spectrum cephalosporins.

CTX-M BL: - Shows greater activity against cefotaxime than other oxymino-BL
(eg CEF, ceftriaxone, cefepime).
- Occurs due to plasmid acquisition of Blasé genes found in Kluyvera
spp.
- Found in different Enterobactericeae.
- Most common ESBL found worldwide.
OXA BLase - Less common, but also plasmid mediated. Amino acid substitution
can also give the ESBL-phenotype.
- Can hydrolyse oxacillin and related anti-staphylococcal penicillin.
- Not all are ESBL, & certain ones only hydrolyses BL such as
penicillin, anti-staphylococcal penicillin & narrow spectrum
cephalosporins.
Others such as - uncommon and found mainly in PSAE. These ESBLs confers high
PER, VEB & resistance to antipseudomonal BL, degrade cephalosporin and
GES monobactams.

- ESBL- isolates typically show greater than average resistance to other agents including
aminoglycosides and fluoroquinoles.

Epidemiology:
- ESBL is reported worldwide and most often isolated from hospital samples as well as
samples from community.
- Prevalence is higher in isolates from Asia, Latin America and middle East.
- Community- acquired ESBL infections have been increasing due to the concomitant high &
increasing rates of fecal colonization by ESBL-producing bacteria worldwide.
- Children are also increasingly affected by this organism.

Transmission:
- Nosocomial transmission occurs due to contamination hands of HCW or contaminated
medical equipment.
- Injudicious use of antibiotics may select for the growth of these organisms.
- Household transmission may occur among family members.
- Environmental, animal and food contamination with ESBL producing gram negative
organism have also been documented which also serves as reservoir for this organism.

Risk factors:
 - GIT is the main reservoir for ESBL producing Enterobactericea, and colonization is a risk
factor for subsequent infection.
- Clinical factors associated with colonization & infection involves healthcare exposure, such
as hospitalization, residence in long term care facility, hemodialysis use & presence of
intravascular catheter.
- RF for community -acquired ESBL includes antibiotic exposure, corticosteroid use and
presence of percutaneous feeding tube.
- Travellers travelling to Asia is a major RF for colonization & infection.
- Travellers’ diarrhea and antibiotic use has been associated with increased risk of acquisition
of ESBL.

Diagnosis:
- Problems with identification arise because ESBL are heterogenous.
- For ex, OXA type are poorly inhibited by clavulanic acid. Some ESBLs are best detected
with CEF and others by cefotaxime.

a) Screening test based on CLSI

Test Comment

Screening - can be performed with 2 methods: broth microdilution or disk diffusion.

test - CLSI screen for ESBL producers is based on MIC (or disc diffusion) cut off for one or
more cephalosporins/aztreonam with:
I, MIC >=2ug/ml for aztreonam, ceftazidime, cefotaxime, ceftriaxone
Ii, MIC >=8ug/ml for cefpodoxime for E. coli, KLPN, K. oxytoca.

- For P. mirabilis,
I, MIC>=2ug/ml for ceftazidime, cefotaxime and cefpodoxime.
KLPN, E. coli, K. oxytoca P. mirabilis
 Cefpodoxime <=17mm Cefpodoxime <=22mm
CEF <=22mm CEF <=22mm
Aztreonam <=27mm Cefotaxime <=27mm
Cefotaxime <=27mm
Ceftriaxone <=25mm

- Due to different levels of expression and hydrolytic activities, the ESBL producing
isolates may produce elevated MICs to one or more screening drugs. Hence, the sensitivity
of screening tests is increased if more than one drug is used for screening.

- Isolates testing positive will need be confirmed with ESBL confirmation test.
Phenotypic - Tests can be performed with either disc diffusion or broth microdilution.
test - Principal is based on addition of clavulanic acid which will enhance the activity of
cephalosporin when tested together compared to its activity when tested alone.
- BROTH MICRODILUTION à cefotaxime and ceftazidime are tested with and without
4microgram clavulanic acid.
- A decrease in MIC >=3 doubling dilution compared to the agents tested alone indicates
positive.
- DISC DIFFUSION à same agents tested in combination with & without 10ug clavulanic
acid.
- An increase in zone diameter >=5mm for either disc with clavulanic acid indicates
positive for ESBL.
** A screen-positive, confirmatory test- negative result may occur:

- isolate may be ESBL negative but demonstrates reduced susceptibility to screening

agents because of decreased porin production, hyperproduction of normal
spectrum beta lactamases, such as TEM1 & SHV1 or production of another
cephalosporinase-hydrolysing enzyme (ex. AmpC enzyme).
- isolate may be ESBL positive but concurrently also produce additional beta
lactamase (ex AmpC enzyme), which is not inhibited by clavulanic acid. (To
improve sensitivity of ESBL detection, add cloxacillin as inhibitor).
- Commercial automated AST systems:
o also used by most labs to identify ESBL based on in-built ESBL screen &
confirmation test on routine testing card.
o These systems overcall the presence of ESBL and some lack FDA approval
for P.mirabilis due to poor performance.
o Labs that report the presence of ESBL using this system may consider
performing additional confirmatory testing for ESBL.

Other techniques:
- Automated system (VITEK, Microscan, BD diagnostics)
- Double disk test in which disc with clavulanic acid is placed near disck with an oxymino-
BL enhances ability to the latter compound.
- E strip with clavulanate added to 1 side of dual oxymino-BL gradient
- pyrosenquencing and microarray
Genotypic: - Commercial assays have been developed for molecular detection of broad-spectrum BL, for
ex Check points and Analytik Jena VYOO.
- Detects ESBL (TEM, SHV, CTX-M), carbapenemases, AmpC & OXA-48.
Advantages Disadvantages
- Short run time and hands on time - Interpretation is challenging.
- can be performed on colonies and direct - Susceptibility of an organism
from specimens, rectal swabs, perirectal cannot necessarily be inferred just
swabs, stools. because no resistant target is
detected.
- no molecular panel is
comprehensive.
- Bacterial strains may possess
multiple BL or non-BL (ex: reduced
outer membrane permeability,
efflux pumps). Therefore,
phenotypic resistance should always
be reported.
- genetic resistance may be detected
even though isolate is
phenotypically susceptible to an
agent for which resistance might
have been predicted.

* How to report discrepant between phenotype and genotype, read CLSI pg 283.
 Treatment (IDSA):
Table 2. Recommended antibiotic treatment options for presumed or confirmed extended-spectrum
β-lactamase producing Enterobacterales (ESBL-E), assuming in vitro susceptibility to agents in table

Source of Antibiotic Rationale

Infection

Cystitis Preferred Treatment - safe and effective

Nitrofurantoin, bactrim

Alternative Treatment - Amoxicillin-clavulanate is an alternative rather because

data have shown it is associated with a higher clinical failure
(First-line options not available
rate than ciprofloxacin for cystitis, presumably due to
or tolerated)
persistent vaginal bacterial colonization.

Amoxicillin-clavulanate, single-
- Aminoglycosides are nearly exclusively eliminated by the
dose aminoglycosides,
renal route in their active form.
fosfomycin (E. coli only)

- Oral Fosfomycin: alternative agent exclusively for

Ciprofloxacin, levofloxacin,
treatment of ESBL-producing E. coli cystitis as
ertapenem, meropenem,
the fosA gene, intrinsic to K. pneumoniae and several other
imipenem-cilastatin
Gram-negative organisms, can hydrolyze the drug and may
lead to clinical failure

Pyelonephriti Ertapenem, meropenem, - these agents are able to achieve high concentrations in the
s or cUTI1 imipenem-cilastatin, urine.
ciprofloxacin, levofloxacin, or
- if carbapenem is first initiated & cipro, levo, Bactrim is
trimethoprim sulfamethoxazole
sensitive, can transition to these drugs.
- Nitrofurantoin and oral fosfomycin do not achieve
adequate concentrations in the renal parenchyma and should
be avoided.

Infections Meropenem, imipenem-cilastatin, - 30-day mortality was reduced for patients with ESBL E.
outside of the ertapenem coli and K. pneumoniae bloodstream infections treated with
meropenem compared to piperacillin-tazobactam.
urinary tract
Oral step-down therapy to - oral step down: these agents are reasonable treatment
ciprofloxacin, levofloxacin, or options, if
trimethoprim sulfamethoxazole (1) susceptibility to the oral agent is demonstrated,
can be considered2. (2) patients are afebrile and hemodynamically stable,
(3) appropriate source control is achieved, and
(4) there are no issues with intestinal absorption.
Treatment (UP TO DATE):
1
Oral step-down therapy can be considered after (1) susceptibility to the oral agent is demonstrated, (2)
patients are afebrile and hemodynamically stable, (3) appropriate source control is achieved, and (4) there
are 2no issues with intestinal absorption.
cUTI: Complicated urinary tract infections are defined as UTIs occurring in association with a structural or
functional abnormality of the genitourinary tract, or any UTI in a male patient.
I, Carbapenems
- preferred treatment for severe infections.
- In absence of resistance and severe infection, ertapenem is an acceptable option & useful in
outpatient settings.
- in bacteremia, meropenem has lower mortality rates compared to tazosin.
- cIAI: ceftolozane-tazobactam, ceftazidime-avibactam & eravacycline (with flagyl for
cephalosporin-BL-inhibitor combo.)
- cUTI: plazomicin (if isolate is resistant to other aminoglycoside), if carbapenems cannot be used &
risk of renal dysfunction.
IV Fosfomycin if carba cannot be used, but not widely available.
- cystitis: oral Fosfomycin or nitrofurantoin.
- no difference in clinical efficacy between mero and imipenem. But meropenem is preferred in cases
of seizures or pregnant patients because of possible CNS toxicity & unknown safety profile in
pregnancy of imipenem.
- Ertapenem has a once daily dosing & good in vitro activity. However, some ESBL strains are
resistant to ertapenem & resistance may develop during therapy. Therefore, it is reserve for
susceptible ESBL producing strain not associated with severe infection.
Ii, Cephalosporin
- not recommended for treatment of severe infections, eventhough organism shows in vitro
susceptibility.
- not encourage to use cefepime as it may lead to treatment failure.
Iii, Tazosin
- not recommended to use for severe infection, but can be used in isolated UTI as alternative
(much higher drug concentration in urine).
Iv, other drugs
- Tigecycline is a potential alternative for treatment, especially for patients with BL allergies.
(Should be avoided for treatment of bacteremia due to low serum concentration, development of
resistance during therapy. But can be given in strain producing carbapenemases in combo with
another drug).
- Eravacycline appears effective but clinical data is limited.
- plazomicin is an advanced aminoglycoside that retains activity against ESBL despite presence of
AME that can inactivate other aminoglycosides.
- Fosfomycin retains activity against ESBL. Oral Fosfomycin can be effective against simple
cystitis.

Prevention:
- *same like mrsa
- Restriction of beta lactam use.
- Discontinuation of inpatient contact can be considered once 6 months have passed since last
positive culture, patient does not have active infection & is not on active treatment of an ESBL
infection
Q8: AmpC & atproducing
least two consecutive negative rectal swabs at least one week apart off antibiotics have
Enterobacterales
been obtained.
- class C serine BL enzymes that can be produced by a no of Enterobacterales & non glucose
fermenter GN bacteria.
- AmpC β-lactamases are cephalosporinases that are poorly inhibited by clavulanic acid and can be
differentiated from ESBLs by their ability to hydrolyze cephamycins.
- Inhibited by boronic acid and cloxacillin.
- AmpC production occurs by one of 3 mechanisms:
 Inducible chromosomal R
 Stable chromosomal de-repression
 Plasmid mediated ampC genes
Mechanism Comment
Inducible chromosomal R - increased AmpC enzyme production resulting from inducible
ampC expression can occur in the presence of specific abx à
results in sufficient enzymes in periplasmic space to increase
MICs
- results in ceftriaxone and ceftazidime resistance.
- Enterobacterales isolate that initially test as susceptible to
ceftriaxone may exhibit non-susceptibility to agent after tx.
Stable chromosomal de- - constitutive ampC production.
repression
- test non-susceptible to ceftriaxone & CEF.
- some Enterobacterales contain mutations in promoters &
attenuators of ampC or other regulartory genes, stably de-
repressing gene expression.
Plasmid mediated ampC genes - test non-susceptible to ceftriaxone & CEF.
- constitutive expression of plasmid borne ampC gene (eg blaCMY,
blaFOX..etc) most commonly observed in eg E.coli, KLPN,
Salmonella
Enterobacterales that should be considered at moderate to high risk for clinically significant AmpC
production due to an inducible ampC gene:

Enterobacter cloacae
Klebsiella aerogenes
Citrobacter freundii

- when above organisms are recovered from clinical cultures (other than those associated with
uncomplicated cystitis), treatment with ceftriaxone & CEF, even if isolates initially test susceptible.
- Serratia marcenscens, M. morganii, Providencia spp are unlikely to overexpress ampC (occurs in
less than 5%). à treatment should be based on susceptibility results.
 When treating infections with high bacterial burden & limited source control, it is
alternatively reasonable to consider tx cefepime instead of ceftriaxone.
 Features to consider when selecting antibiotics for infections caused by organism with moderate to
high risk of clinically significant inducible AmpC production.
- both the ability to induce ampC genes & inability to withstand AmpC hydrolysis should inform
abx decision making.
- Potent AmpC inducers: aminopenicillins (amoxi, ampi), narrow spectrum (ie 1st gen)
cephalosporins, cephaycins à test non-susceptible
- Imipenem are also potent ampC inducers but resistant to AmpC-E hydrolysis.
- Mero & erta also similar with imipenem.
- tazosin, ceftriaxone, ceftazidime & aztreonam à weak ampC inducers
 ceftriaxone & ceftazidime are susceptible to hydrolysis à not effective for tx.
 cefepime is a weak inducer of ampC but also can withstand hydrolysis à effective agent
 Tazosin à susceptible to hydrolysis
- fluroquinolones, aminoglycoside, Bactrim, tetracycline or other non-BL do not induce ampC and not
hydrolysed by ampC.

Role of antibiotics in treatment of moderate to high risk of clinically significant inducible AmpC
production:
Antibiotics Rationale/Role
Cefepime - suggested treatment when Cefepime MIC <= 2mcg/mL
- If MIC >=4mcg/mL à CARBAPENEM is suggested (assuming it is S as ESBL co-
production may be present). (ie ertapenem, meropenem, imipenem-cilastatin).
- if there’s clinical failure, need to think of ESBL co-producer, presence of outer membrane
porin mutations.
- important to note that cefepime is not effective against ESBL-E.

Ceftriaxone - not recommended for invasive infections

- reasonable tx option for uncomplicated cystitis when susceptibility is demonstrated.

Tazosin - not suggested for treatment of serious infections.

- tazobactam less effective at inhibiting AmpC hydrolysis
- reasonable tx option for uncomplicated cystitis

BL-BLI & BL - BL à cefiderocol

- BL-BLIà ceftazidime-avibactam, imipenem-cilastatin-relebactam, meropenem-
varbobactam
- increased potency against AmpC, but preferentially reserved for treating infection caused
by carbapenem resistance organism.
- panel does not suggest use of ceftolozane- tazobactam (reason point 2 above)

Non-BL - Bactrim/flouroquinolones can be considered for the tx of INVASIVE infections, either IV

or as oral step-down, as dictated by clinical status, likely source of infection, ability to
consume & absorb oral abx, after abx susceptibility is demonstrated.
- uncomplicated cystitis: nitrofurantoin, Bactrim or single IV dose of aminoglycoside

How to diagnose AmpC: (examples taken from seminar)

- Currently no CLSI guideline for detection
- Disk diffusion using FOX has poor specificity: of 45 isolates with FOX resistance, 64% were non
ampC producer (Coudron et al 2000)
- Need to confirm with further test
Method Principle Interpretation
Disc Potentiation - Performed on all non ESBLs - An increase in zone by ≥ 5mm around the disc with
Test (DPT) isolates using three different inhibitor compared to the zone formed by the disc
enzymes inhibitors, i.e., boronic acid without the inhibitor, confirmed the test organism to be
400µg (10µl), cloxacillin 300µg an AmpC producer as the inhibitor potentiated the
(10µl) and 500µg (10µl). inhibitory effect of the cephalosporins by inactivating
the AmpC enzyme.
- each of these substances by
inactivating the AmpC enzyme
produced by the organism,
potentiate the inhibitory effect of the
antibiotic on the organism.
- test was performed separately for
each inhibitor.
- A lawn culture of the test
organisms was made.
- Two discs each of ceftazidime
(30µg) and cefotaxime (30µg) were
placed on the lawn culture distant
(25 mm) from each other.
- A 10µl of freshly prepared enzyme
inhibitor under test was added to one
of the two disc of each
cephalosporin and the zone of
inhibition was measured.
Double Disc - uses boronic acid 400 µg (10µl), After overnight incubation at 37 C, expansion of
Synergy Test cloxacillin 300(10µl) µg & 500 µg inhibitory zone of either one or both, ceftazidime and
(DDST) (10µl) cefotaxime, towards the inhibitory discs was
interpretated as positive results for production of
- Using MH plate for each test
AmpC β lactamases by the isolates.
organism, a lawn culture was made,
the disc of the inhibitor was placed
in the centre and the distance
between this disc and cefotaxime
(30µg) and ceftazidime (30µg), one
on either side from centre to centre,
was kept as 15 mm.

AmpC disk - A lawn culture of a 0.5 - After an overnight incubation at 37°C, the plates
McFarland’s suspension of were examined for either an indentation or a flattening
ATCC E. coli 25922 was prepared of the zone of inhibition, which indicated the enzyme
 on a Mueller-Hinton agar plate. inactivation of cefoxitin (positive result), or an absence
of distortion, which indicated no significant
- A 30 μg cephoxitin disc was
inactivation of cefoxitin (negative result).
placed on the inoculated surface of
the agar.
- The test is based on use of Tris-
EDTA to permeabilize a bacterial
cell wall and release β-lactamases
into the external environment.
- AmpC disks (i.e., filter paper disks
containing Tris-EDTA) (6mm)
which was inoculated with several
colonies of the test organism was
placed beside the cephoxitin disc,
almost touching it.
Modified Hodge - Using cefoxitin disk. - If the test organism expresses AmpC, it hydrolyzes
Test/cephamycin- the cefoxitin and shows growth along the intersection
-Test organism is streaked toward
Hodge test of the streak and the zone of inhibition from the
the cefoxitin disk.
cefoxitin disk.

Etest AmpC - comprises a strip impregnated with - Ratios of cefotetan versus cefotetan/cloxacillin of ≥8
a concentration gradient of cefotetan was considered positive
on one half of the strip and cefotetan
with cloxacillin on the other half of
the strip.
- MICs of cefotetan alone and
cefotetan with cloxacillin were
determined as recommended by the
manufacturer.
Molecular 1) AmpC promoter sequencing (E. 2) Detection of plasmid-mediated ampC beta-
coli only). lactamase genes.
- The ampC promoter sequences - For the detection of plasmid-mediated ampC beta-
were compared to the wild- lactamase genes, a multiplex PCR was used which
type ampC sequence of E. coli strain detects the six plasmid-mediated ampC families (CMY
ATCC 25922. homologues was the most predominant gene (86.9%)
followed by DHA (21.7%), FOX (17.3%), EBC
(13%), and MOX (13%) (Helmy & Wasfi, 2014).
Detection of ESBL in organism that also produce ampC
- ESBL detection may be masked by high-level production of AmpC.
- Strains with the co-existing AmpC β-lactamases may give false negative tests for the detection of
ESBLs, because AmpC β-lactamases are resistant to β-lactamase inhibitors like clavulanic acid and
hence the augmentation in zone diameter in DDST by ESBL producers can be completely masked by
AmpC enzymes.

- Clavulanic acid which is used in the standard DDST test for the ESBL detection acts as an inducer
of the high level AmpC production and it leads to the resistance to the 3rd generation cephalosporins
as well as to the 3rd generation cephalosporins + clavulanic acid.

- So, even if ESBL is present, it will not be detected and it may result in a false negative test

Modified double disk synergy test (used in USM)

- original DDST was modified for detecting ESBLs in AmpC co-producers by placing a disc of
piperacillin –tazobactam at a distance ranging between 22 and 25mm from cefepime disc.
- A disc of augmentin was also placed on the surface of MHA with discs of cefpodoxime, ceftazidime,
cefotaxime, and cefepime at a distance ranging between 16 and 20 mm from it.
- Modification of double disc synergy tests that combine piperacillin-tazobactum with cefepime
enhances the possibility of ESBL detection.
- MDDST showing synergism of only cefepime (indicator drug) but none of the 3 GC used with
amoxicillin-clavulanate. (AMC–Amoxyclav, CTR-Ceftriaxone, CTX-Cefotaxime, CPD-
Cefpodoxime, CPM- cefipime) should be further tested for the AmpC enzyme production.
Q5: CRE
Definition of CRE (CDC):
Any E. coli, KLPN, K. oxytoca, K. aerogenes & Enterobacter spp that test resistant to
meropenem, imipenem, ertapenem, doripenem, meropenem/varbobactam & imipenem/relebactam
with standard susceptibility methods (especially MIC>4mcg/ml for meropenem, imipenem,
doripenem, meropenem/varbocatam, imipenem/relebactam or >2mcg/ml for ertapenem)
OR production of carbapenemase (specifically NDM, VIM, IMP, KPC, OXA-48) demonstrated
with validated molecular methods (ex PCR, CarbaNP, modified hodge test).
Mechanism of resistant:
3 major mechanisms, which are enzyme production, porin mutation & efflux pump.
- Main mechanism is the enzyme production.
- Divided into 2 subgroups:
- CP-CRE (carbapenemase producing)
- Non-CP-CRE (non carbapenamase producing CRE)
Class A - characterised by hydrolysing activity that requires serine active site.
- includes narrow spectrum BBL (penicillinase) & ESBL (cephalosporinase) in TEM, SHV,
CTX-M & BL that possess carbapenamse activity.
- Class A with carbapenemase activity encoded in plasmid or chromosome.
- Chromosomally encoded carbapenemase includes SME, IMI, NMC while plasmid encoded
carbapenemase includes KPC.
- KPC enzyme reside in transmissible plasmid & confer resistance to most BL and can be
transferred from KLPN to other bacteria, such as E. coli, PSAE, Citrobacter, Salmonella,
Shigella etc.

Class B - aka MBLs, because it needs zinc for effective hydrolysis.

- As a result, it can be inhibited by EDTA (iron chelator).
- However, it is not inhibited by BL inhibitor such as clavulanic acid, sulbactam, tazobactam
& avibactam.
- natural occurring or acquired MBLs.
- Naturally occurring are chromosomally encoded & can be found on A. hydrophilia, STMA,
Chryseobacterium spp.
- Acquired MBLs are encoded in integrons residing in plasmid & are transmissible between
species & genera.
- usually multidrug resistance, often co-expressing ESBLs, which inactivate monobactam.

Class D - AKA as OXA-type because of its ability to hydrolyze oxacillin (rather than penicillin)
- affected by BL inhibitor clavulanate, sulbactam and tazobactam.
- There can be transmissible through plasmid (ex OXA-48, OXA-23, OXA-58) or
chromosomally encoded (ex OXA-51).
- most widespread is the oxa-48.

Non- - includes production of beta lactamases, porin loss or overexpression of efflux pump.
carbapenemase - mechanism can appear among themselves or paired with carbapenemase production.
producing: - Enterobactericea can produce different type of beta lactamase, ex AmpC BL, which does
not degrade carbapenem but form bonds with it to prevent it from assessing target site.
- Resistant-nodulation-division (RND) efflux pump are a major mechanism for
Enterobactericea & the resistant genes can be easily transmitted through plasmid.
Diagnosis
a) CP-CRE:
Based on CLSI
- Carbapenemase-producing isolates of Enterobacterales usually test intermediate or resistant to one
or more carbapenems using the current breakpoints.
* Testing not susceptible to ertapenem is often the most sensitive indicator of carbapenemase
production and usually test resistant to one or more agents in cephalosporin subclass III (eg,
cefoperazone, cefotaxime, ceftazidime, ceftizoxime, and ceftriaxone).
3 phenotypic tests approved by CLSI for epidemiological & infection prevention purposes:
CarbaNP, eCIM, mCIM, others Modified Hodge Test (MHT)(no endorsed by CLSI)
Method Principle Advantages Limitations Results

1. MHT - not endorsed by - sensitivity 95% - may have false

CLSI & specificity 91% positive result
with ESBL/AmpC
- involves streaking a - easy to perform
isolates with porin
line away from disk
- cheap defect.
impregnated with
either - Limitation in
meropenem/imipenem detection of NDM-
which was previously producing
streaked on agar plate Enterobacterales
inoculated with lawn (class B).
of carba sensitive
- Time consuming,
E.coli strain.
challenging to
- MHT relies on interpret.
carbapenamase
- Requires
producer to reduce the
overnight
local concetration of
incubation.
carba
antibioticàenable
carba S E.coli to grow
uninhibited around
streak lineà cloverleaf
appearance =>positive
production of
carbapenemase.

2. CarbaNP - Based on hydrolysis - TAT on SAME - Reduced

method. day sensitivity for
detection of OXA-
- Measures in vitro - high level of
48-like
hydrolysis of sensitivity (>
carbapenemase
imipenem in bacterial 90%) and
(class D), which
extracts & produces specificity (>
may lead to FALSE
colour changes within 90%) in detecting
 2 hours. KPC, NDM, NEGATIVE result.
VIM, IMP, SPM,
- Imipenem hydrolysis - Various
and SME-type
results in carboxylic interpretation of
carbapenemases
derivative which results
reduces pH & shifts - in detection of
- Mucoid colonies
colour of phenol red NON-LACTOSE
may also give
indicator from red to FERMENTERS:
FALSE
yellow.
PSAE: sensitivity NEGATIVE result
98%, specificity
- Need to prepare
98%
reagent frequently
due to short shelf
life.
- in detection of
NON-LACTOSE
FERMENTERS:
ACBA:
2 microcentrifuge tube for sensitivity
19%,
each patients, QC &
specificity
uninoculated reagent tubes.
100%
- oxa type
carbapenemase
less effective in
hydrolysing
beta lactam
ring of
antitbiotic
- resistant
mechanism
other than
carba
productionà
porin
expression,
upregulation of
efflux pump.
- not
recommended
for use in
ACBA.
3. Carbapenem Inactivation - Incubate - Sensitivity 91%, - Less sensitive in
Method 10microgram of specificity 94% detection of OXA-
meropenem disc in type
- Less costly
water with 10microL carbapenamase,
compared to
loop of carbapenem MBL/ in low levels
CarbaNP
producing isolate for 2 of carbapenemase
 hours. - Performance & expression.
interpretation is
- if meropenem not straightforward
hydrolysed, isolate
does not produce
carbapenemase.

- after incubation,
meropenem disc is
removed & placed on
MHA lawn with
susceptible E. coli
strain & incubate
overnight.

- zone of inhibition is
measured the next day.
a) mCIM (modified Carbapenem * Steps above is - Sensitivity > 99% - Investigations of POSITIVE
Inactivation method)- modified by preparing and specificity > mCIM with 15mm zone
bacterial suspension in 99% for detection Acinetobacter spp. presence of
Enterobacterales & PSAE pinpoint
tryptic soy broth & of KPC, NDM, showed poor
incubate 4 hours VIM, IMP, IMI, specificity and poor colonies wi
a 16–18 mm
SPM, SME and reproducibility
zone
OXA-type between laboratories, * If merop
carbapenemases and performing hydrolysed
among mCIM with inhibition o
Enterobacterales Acinetobacter spp. is limited gro
isolates. not endorsed by inhibition
CLSI.
- Sensitivity > 97% NEGATIV
and specificity - False positive may >=19mm (c
100% for detection occur in AmpC zone)
of KPC, NDM, hyperproducing
VIM, IMP, IMI, E.clocae. * if isolate
SPM and OXA- not produce
- Needs overnight carbapenam
type
incubation. mero not
carbapenemases
among - CANNOT hydrolyse,
DIFFERENTIATE growth not
P. aeruginosa inhibited.
BETWEEN
isolates.
SERINE (class A) & INTERME
MBL (class B). TE: 16-18m
OR >=19m
but with
presence of
points with
the zone. (T
presence or
absence of
carbapenem
cannot be
confirmed.
b) eCIM * Add 20microL of 0.5 - Sensitivity > - If co-producing - If mCIM
M EDTA in TSB in 95% and serine & MBLà POSITIVE
carbapenem specificity > 92% false negative 18mm
inactivation step, for diameter),
bacteria and differentiation of interpret
meropenem disc, metallo-β- eCIM.
incubate 4 hours and lactamases
-
then placed on MHA (NDM, VIM, and
Interpretat
lawned with E. coli IMP) from serine
ATCC. carbapenemases >= 5mm
(KPC, OXA, and increase i
* Test should be
SME) among zone diam
performed together
Enterobacterales of eCIM
with mCIM.
isolate. compared
- If isolate is an MBL mCIM
producer, EDTA will suggests M
inhibit carbapenamse producing
productionà strain.
meropenem disc is not
hydrolyzedà E. coli
ATCC growth is
inhibited.

4. Lateral flow assay - antibody-based - able to detect - only able to detect - Positive
method to identify VIM, NDM, among results bas
presence of KPC, OXA-48 Enterobacterales on the
carbapenamases. carbapenamses in presence o
- requires colony
5mins directly visible lin
- TAT same day (overnight growth).
from cultured specific fo
bacteria - false-positive carbapene
results with some -type
-For Enterobacteri
non carbapenemase
aceae, sensitivity,
OXA-enzymes
100% and
specificity, ≥95%
- Easy to perform,
no special
reagents or media
necessary besides
the test kit
5. Genotypic method - freshly prepared - - Complex to
 Maldi-TOF cultures are mixed w Enterobacteriacea interpret; requires
carbapenem solutions e, PSAE, ACBA MALDI-TOF
such as ertapenem/ instrument settings
-For Enterobacteri
mero & incubated for different from those
aceae,
2-4hr at 35-37c. traditionally used
sensitivities,
for FDA-approved
- Mixture is then 77%–100% and
microbial
centrifuged & the mass specificities,
identification
spectrometry 94%–100%
techinique is used to
- same day
measure supernatant.
results, 3-4hrs
- in case of
carbapenem
hydrolysis, the
degradation products
& sodium salt of the
carbapenem molecule
is visible in spectra.
PCR
- performed in IMR, using gel electrophoresis.
- Detects IMP, NDM, KPC,

Treatment:
 Table 3. Recommended antibiotic treatment options for carbapenem-resistant Enterobacterales
(CRE), assuming in vitro susceptibility to agents in table

Source of Antibiotics Rationale

Infection
Cystitis Preferred Treatment - achieve high concentrations in urine.
Ciprofloxacin, levofloxacin, - Aminoglycosides: exclusively eliminated by the renal
trimethoprim route in their active form. A single intravenous dose is
sulfamethoxazole, effective with minimal toxicity.
nitrofurantoin, or a single-
dose of an aminoglycoside - Meropenem should be avoided if carbapenemase
testing is positive, even if susceptibility to meropenem is
demonstrated.
Meropenem1 (standard-
infusion): only if ertapenem
resistant, meropenem
susceptible, AND
carbapenemase testing
results are either not
available or negative.
Alternative Treatment - Colistin converts to its active form in the urinary tract;
(First-line options not but theres a risk of nephrotoxity.
available or tolerated)
- Polymyxin B should not be used due to its
Ceftazidime-avibactam, predominantly nonrenal clearance.
meropenem-vaborbactam,
imipenem-cilastatin-
relebactam, and cefiderocol

Colistin (only when no

alternative options are
available)
Pyelonephritis Preferred - Extended-infusion meropenem is a preferred agent
or cUTI2 Ceftazidime-avibactam, against pyelonephritis and cUTI by CRE that remain
meropenem-vaborbactam, susceptible to meropenem, since most of these isolates
imipenem-cilastatin do not produce carbapenemases, but should be avoided
relebactam, and cefiderocol if carbapenemase detected
Meropenem1 (extended-
infusion): only if ertapenem
resistant, meropenem
susceptible, AND
carbapenemase testing
results are either not
available or negative.
 Alternative
Once-daily aminoglycosides

Infections Preferred - Extended-infusion meropenem is recommended

outside of the Meropenem1 (extended- against infections outside of the urinary tract by CRE
urinary tract infusion) that remain susceptible to meropenem since most of
these isolates do not produce carbapenemases but should
Resistant to
be avoided if carbapenemase detected
ertapenem,
susceptible to
meropenem,
Alternative - Reserve ceftazidime-avibactam for treatment of
AND
carbapenemase Ceftazidime-avibactam infections caused by CRE resistant to all carbapenems,
testing results are to preserve its activity.
either not
available or
negative

Infections Preferred - agents are associated with improved clinical

outside of the Ceftazidime-avibactam, outcomes and reduced toxicity compared to other
urinary tract meropenem vaborbactam, regimens
and imipenem-cilastatin
Resistant to
relebactam
ertapenem,
meropenem, Alternative
AND Cefiderocol - an alternative treatment option for CRE infections,
carbapenemase regardless of the mechanism of resistance to
testing results are carbapenems.
either not
available or Tigecycline, eravacycline
negative (intraabdominal - acceptable monotherapy options; high dose
infections) tigecycline may be more effective than standard dose
tigecycline.
- limited to the treatment of intra-abdominal infections.
- These agents achieve rapid tissue distribution
following administration, resulting in limited
concentration in the urine and poor serum
concentrations
KPC identified Preferred
Ceftazidime-avibactam,
(Or meropenem-
carbapenemase vaborbactam, imipenem-
positive but cilastatin relebactam
identity of
carbapenemase Alternative
unknown3) Cefiderocol

Tigecycline,
eravacycline
(intraabdominal
infections)
Metallo-β- Preferred
lactamase (i.e., Ceftazidime-avibactam +
NDM, VIM, or aztreonam, cefiderocol
IMP)
carbapenemase Alternative
identified Tigecycline, eravacycline
(intraabdominal infections)

OXA-48-like Preferred - Meropenem-vaborbactam and imipenem-cilastatin-

carbapenemase Ceftazidime-avibactam relebactam have limited to no activity against CRE
identified producing OXA-48-like enzymes.

Alternative
Cefiderocol

Tigecycline, eravacycline
(intraabdominal infections)
1
The majority of infections caused by CRE resistant to ertapenem but susceptible to meropenem
are caused by organisms that do not produce carbapenemases.
2
cUTI: Complicated urinary tract infections are defined as UTIs occurring in association with a
structural or functional abnormality of the genitourinary tract, or any UTI in a male patient.
3
The vast majority of carbapenemase producing Enterobacterales infections in the United States
are due to bacteria that produce Klebsiella pneumoniae carbapenemase (KPC). If a disease-
causing Enterobacterales is carbapenemase-producing but the specific carbapenemase enzyme is
unknown, it is reasonable to treat as if the strain is a KPC-producer. If a patient is infected with a
CRE strain with an unknown carbapenemase status and the patient has recently traveled from an
area where metallo-β-lactamases are endemic (e.g., Middle East, South Asia, Mediterranean),
treatment with ceftazidime-avibactam plus aztreonam, or cefiderocol monotherapy are
recommended. Preferred treatment approaches for infections caused by metallo-βlactamase
producers also provide activity against KPC and OXA-48-like enzymes.
 Antibiotic regime Rationale

Role of polymyxins? - Increased mortality and excess nephrotoxicity associated with

polymyxin-based regimens.
- Clinical and PK/PD data suggest
Polymyxin B and
that these agents have limited clinical efficacy, so CLSI
colistin should be
eliminated susceptibility category.
avoided for the
treatment of infections - should be avoided, with the exception of colistin as a last
caused by CRE. resort agent against CRE cystitis.
Colistin can be
- Polymyxin B should not be used as treatment for CRE cystitis, due
considered as a last
to its predominantly nonrenal clearance.
resort for
uncomplicated CRE
cystitis
Role of combination - combination does not offer any value.
therapy?
- continued use of a second agent increases the likelihood of
antibiotic-associated adverse events
Combination
antibiotic therapy (i.e.,
the use of a β-lactam
agent in combination
with an
aminoglycoside,
fluoroquinolone, or
polymyxin)
Q6: VRE
Definition CDC
Enterococcus faecalis, Enterococcus faecium, or Enterococcus species unspecified (only those not
identified to the species level) that is resistant to vancomycin, by standard susceptibility testing
methods or a laboratory finding of VRE (includes but not limited to PCR or other molecular based
detection methods).
Mechanism of glycopeptide resistance:
- Vancomycin inhibits enterococci by binding to the D-alanyl-D-alanine (D-Ala-D-Ala) of the
peptidoglycan, inhibiting cross linking of cell wall by transpeptidation.
- main mechanism of glycopeptide resistance (e.g., vancomycin) in enterococci involves the
alteration of the peptidoglycan synthesis pathway, specifically the substitution of D-Alanine-D-
Alanine (D-Ala-D-Ala), to either D-Alanine-D-Lactate (D-Ala-D-Lac) or D- Alanine-D-Serine (D-
Ala-DSer) -à leads to less binding affinity of glycopeptide drugs.
- Definition of VRE: Vancomycin MIC >=32 (S: <=4, I: 8-16).
a) Intrinsic - most significant resistance is resistance to aminoglycosides, beta lactamase-induced
resistance: ampicillin resistance and glycopeptides.
-* Resistance to high-level beta-lactams (MIC 16-64 µg/mL) is due to mutation or
overproduction of penicillin binding protein 5 (PBP5).
- Resistance to low level aminoglycosides (MIC 62 to 500 µg/ mL) is because of slow
uptake.
b) Acquired - Mutation in the existing DNA or acquisition of foreign DNA results in acquired resistance.
resistance:
- High-level aminoglycoside resistance (MIC >2000 µg /mL), either ribosomal mutation is
acquired or aminoglycoside modifying enzyme is acquired via plasmid.
- Resistance to chloramphenicolà either enzymatic or plasmid-borne
- Resistance to high-level erythromycin à transposon encoding macrolide resistance.
- Resistance to glycopeptides à plasmid-borne or chromosomal and is due to a cluster of
genes absent in normal enterococci.
Factors of emergence of VRE:
 Prolonged hospitalisation, younger age, use of ceftriaxone and vancomycin.
 Hospital workers can also transmit VRE as it can survive on fingers for about 30
minutes even after washing hands.
 Companion animals and pets can also be a reservoir for VRE.
Phenotypic classification:
- 9 types of resistance genes have been identified: VanA, VanB, VanC, VanD, VanE, VanG, VanL,
VanM and VanN.
- genes confer high to moderate level resistance to vancomycin and teicoplanin.
- VanA & VanB are the most prevalent.
Phenotype Level of resistance in Vancomycin & Teicoplanin Types
Van A High level resistance E. fecalis
vancomycin (MICs > 64 µg/mL) E. feacium
teicoplanin (MICs >16 µg/mL)
Van B modest levels of vancomycin (MICs 32 to 64 µg/mL) (CDC: 16-64) to high E. fecalis
levels of vancomycin (4 to >1,000 µg/mL) but show susceptibility towards
E. feacium
teicoplanin (<=1)
Van C Low level of resistance, Vancomycin MIC 2-16 E. gallinarum
E. casseliflavus
Van D vancomycin (MICs 64 to 128 µg/ml), teicoplanin (MICs 4-8 µg/ml) E. feacium
Treatment in general:
*Antimicrobial therapy for enterococcal bacteremia is warranted in the setting of:
- two or more positive blood cultures
- a single positive blood culture accompanied by signs of sepsis
- a single positive blood culture together with a positive enterococcal culture from another usually
sterile site.
Antibiotic Notes
Linezolid Bacteriostatic, approved for used.
Binds to 50S ribosome, preventing peptide bond formation and thus the addition of new
amino acids.
High bioavailability after oral administration, and it achieves therapeutic levels in most
tissues.
Daptomycin bactericidal cyclic lipopeptide antibiotic that causes depolarization of the bacterial cell
membrane.
Approved for clinical use in vancomycin sensitive
Oritavancin Semisynthetic glycopeptide that inhibits cell wall synthesis and has in vitro bactericidal
activity against staphylococci and enterococci, including VRE
Tigecycline Glycylcycline antibiotic derived from minocycline with in vitro bacteriostatic activity
against many gram-positive pathogens (including methicillin-resistant S.
aureus [MRSA], VRE, and penicillin-resistant Streptococcus pneumoniae), some gram
negatives (important exceptions include Pseudomonas, Proteus, Providencia,
and Morganella species), anaerobes, and atypical species.
Not used for VRE, may be used in vanco S Enterococci
Quinupristin- Streptogramin antibiotics with in vitro activity against VRE, withdrawn for use.
dalfopristin
Poor activity against E. faecalis due to a species-specific adenosine triphosphate (ATP)-
binding protein
Teicoplanin Glycopeptide which has in vitro activity against E. gallinarum and E.
casseliflavus (VanC VRE) as well as most VanB-type VRE, although it is rarely active
against VanA-type VRE.
Televacin Lipoglycopeptide approved for complicated skin and skin structure infections caused by
susceptible gram-positive bacteria, including S. aureus and vancomycin-susceptible E.
faecalis.
Infection prevention & control:
- vanA and vanB genes are transferable and can spread from organism to organism.
- vanC genes are not transferable, have been associated less commonly with serious infections, and
have not been associated with outbreaks.
General principles: (same for all, the only difference is the sample for surveillance)
- Hand hygiene
- Contact precautions: Wearing gown and gloves when entering a patient room and removing them
prior to exiting may decrease VRE transmission.
- The optimal duration of contact precautions for VRE is uncertain and clinical practice is variable.
- SHEA guidelines: discontinuation of contact precautions after documentation of one to three
negative weekly stool or rectal swab surveillance cultures and state that that extension of contact
precautions is reasonable for patients who are highly immunosuppressed, being treated with broad
spectrum antibiotic therapy without VRE activity, or receiving care in protected environments (such
as burn units) or in institutions with high VRE rates.
- VRE colonization may be prolonged, with high relapse rates after multiple consecutive negative
surveillance cultures.
- UP to DATE: - During the first year following VRE infection, we favor documentation of three
negative weekly stool or rectal swabs discontinue contact precautions;
- if more than one year has passed since VRE infection, we favor one negative stool
or rectal swab.
- Decolonization: no effective methods. Treatment specifically for this is not recommended.
- Surveillance culture: SAMPLES: Rectal swabs, perirectal swabs, or stool samples, areas of wound
breakdown and draining wounds.
- Surveillance cultures are not needed from patients known to have been
colonized or infected with VRE within the past 6 to 12 months ---> remain
culture positive regardless of therapy.
- Clinical cultures should not be used as a surrogate for VRE surveillance
cultures due to a lack of correlation.
- Active surveillance of high-risk patients: Reduces transmission of VRE when performed in
outbreak settings or in high-risk patient units such as ICUs and hematology-oncology wards.
- Enviromental disinfection: Improving room disinfection through education and performance
feedback for environmental services
personnel. Enhanced disinfection strategies such as vaporized
hydrogen peroxide and ultraviolet (UV) light.
- Patient bathing: Chlorhexidine bath.

Q7: MDR/XDR/ Pan-DR Acinetobacter spp

Definitions (CDC):

Main mechanisms of resistance

Enzymatic mechanism of resistant
1. OXA-type carbapenemases
- Class D (oxallinase) hydrolyses oxacillin and has a serine in the active catalytic site.
- OXA enzymes such as OXA-23, OXA-24/40, OXA-58, OXA-143 and OXA-235 are among the
most prevalent in A. baumannii.
- Genes encoding OXA-type β-lactamases have been identified mainly chromosomally or plasmid
located in A. baumannii strains.
- From Prof Zak, in m’sia, OXA-23 and OXA-51 are more prevalent.

2. Aminoglycoside modifying enzymes

- Enzymatic modification of AGs through production of aminoglycoside-modifying enzymes (AMEs)
is the main mechanism of resistance in A. baumannii.
- classified into acetyltransferases, phosphotransferases and nucleotidyl transferases.
- Other resistance mechanisms are 16S ribosomal methylases.
Non-enzymatic mechanism of resistant
1. Activation of the efflux pump
- encoded by chromosomal genes responsible for resistance to several antimicrobial agents in case of
overexpression.
- four categories of efflux pumps: RND superfamily (resistance–nodulation–division superfamily),
MATE (multidrug and toxic compound extrusion family), MFS (major facilitator superfamily) and
SMR (small multidrug resistance transporters).
- RND system is more represented in A. baumannii, which includes the AdeABC pump, with an
essential role in resistance to antimicrobial agents, especially aminoglycosides.

2. Decreased membrane permeability

- pores of the outer membrane have an essential role in the resistance and virulence of A. baumannii
strains, by mediating the transport of the molecules.
- decreased membrane porin density (Omp22–23, Omp43, Omp44, Omp47, Omp33–36, Omp37 and
CarO) is associated with increased carbapenem resistance
- also OmpA à association of this porin with aztreonam, chloramphenicol and nalidixic acid
resistance.

3. Changing the target site

- this mechanism is based on random point mutations that have a minimal impact on bacterial cell
homeostasis
- Rifampicin resistance:
 point mutations is rifampin resistance (RIF).
 Rifampin blocks RNA polymerase activity. The region to which rifampin binds is a
highly conserved enzymatic structure in the β subunit of RNA polymerase encoded
by the rpoB gene.
 After the attachment to the binding site, the antibiotic molecule blocks the
transcription by inhibiting nascent RNA
 Rifampin resistance can also be mediated by the arr-2 gene encoding an ADP-
ribosyltransferase, found within class 1 integrons
- Fluroquinolone resistance:
 most common fluoroquinolone resistance mechanism is represented by the
spontaneous mutations in the gyrA, gyrB and parC genes encoding gyrase and
topoisomerase IV.
 also mediated by plasmid-mediated quinolone-resistance genes (PMQR), either
through the QNR protein, a pentapeptide that protects target enzymes from antibiotic
action or through an aminoglycoside-modifying mutant enzyme

- Colistin resistance:
  i) modification of the lipid A from LPS by mutations in the PmrAB two-component
system and
 ii) loss of LPS production capacity due to mutations in the lpxA, lpxC and lpxD
genes.
* Gene encoding colistin R -à MCR-1 gene (can be found in Enterobactericeae, not sure about
ACBA)

Clinical presentation:
- most frequent presentation: VAP and blood stream infection.
- Acinetobacter can colonize skin, wound, GIT and respiratory tract.
- difficult to distinguished between colonization and true infection and most infection preceded by
colonization.
Clinical Comments
manifestation
HAP - occurs predominantly in ICU.
- patients who require mechanical ventilation and tends to be characterized by a
late onset.
- most cases occur in previously colonized patientsà True Acinetobacter
pneumoniae must be distinguished from airway colonization in mechanically
ventilated patients.
CAP - fulminant illness with abrupt onset and rapid progression to respiratory failure
and hemodynamic instability.
BSI - most frequent sources are vascular catheters and the respiratory tract.
- Less common primary sites include wounds and the urinary tract.
- Risk factors include intensive care, mechanical ventilation, prior surgery, prior
use of broad-spectrum antibiotics, immunosuppression, trauma, burns,
malignancy, central venous catheters, invasive procedures, and prolonged hospital
stay
SSTI - may contaminate surgical and traumatic wounds, leading to severe soft tissue
infection that can also progress to osteomyelitis.
- Surgical wound infections are frequently related to the presence of prosthetic
material
UTI - urinary tract can become colonized readily with Acinetobacter, particularly in
the setting of indwelling urinary catheters; the incidence of infection is low.
- In the absence of other signs or symptoms of infection, isolation
of Acinetobacter may be attributed to colonization.

* Endocarditis and meningitis caused by Acinetobacter are rare.

IDSA guideline for the treatment of CRAB

Management is difficult due to:
- most commonly recovered from respiratory specimen or wounds à isolate may represent coloniser
or true pathogen.
- once it exhibits carbapenem resistance, it generally has acquired resistance to most other antibiotics
expected to be active against wild-typeàlimited antibiotic option.
- production of carbapenemases eg OXA-24/40-like & OXA-23 mediates resistance to carbapenems
- May also produce MBL & serine carbapenemases -àfurther limits options.
- Sulbactam resistance appears to be driven primarily via mutations targeting PBP
- Quinolone resistance à due to mutations in chromosomally encoded quinolone resistance
determining regions upregulate efflux pump.

General approach to treatment:

Severity of Treatment
infection
Mild - unasyn as preferred agent (single agent).
- UTI, skin & soft tissue, tracheitis, with clinical changes indicating infection (not
colonization), BUT without hemodynamic instability.
- favours high dose unaysn monotherapy, but standard dosing is reasonable, if intolerance
or toxicities precludes the use high dosage.
- Alternative: minocycline, tigecycline, polymyxin B (colistin for cystitis) or cifederocol.
- if non-susceptible to unasyn is demonstrated, high dose unasyn may still remain an
effective option.
- Second active agent can be added if CRAB is not susceptible to unasyn.
Moderate to - combination with at least 2 agents with in vitro activity.
severe - High dose unasyn is suggested as a component of combination therapy.
- Step-down therapy can be considered after clinical improvement can be demonstrated.
- Rationale for combo therapy:
 Lack of robust clinical data supporting tx with single agent demonstrating
in vitro activity.
 High bacterial burdens due to universal delay in initiating effective
therapy (common empirical tx not effective against CRAB)
 Susceptible patients are generally chronically & critically ill with
potential impaired immune systems & the use of combo therapy may
hasten recovery.
 Abx that initially appear active may rapidly develop resistance. Combo
therapy increases the likelihood that at least one active agent is being
administered.
- potential options for combo: unasyn (preferred), tetracycline derivatives (minocycline,
followed by tigecycline), polyB, extended- infusion meropenem or cefiderocol.
- unasyn should be still be included in combo therapy even though it demonstrates
resistance.
- combo of meropenem & colistin (or polyB), without third agent, is not recommended à
can however, consider combo of unasyn, meropenem, polyB.
Role of specific antibiotics in treatment of CRAB
 Antibiotic Rationale
Unasyn - high dose unaysn preferred, regardless of whether in vitro activity has been
demonstrated.
- Sulbactam is a competitive, irreversible BLI that in high doses saturates PBPs
(PBP1 & 3).
- Ampicillin – sulbactam uses 2:1 formulation,
EX: 3g of unasyn comoprises of 2g of ampicillin and 1 g of sulbactam.
Total daily dosage of 27g is equivalent to 9g of sulbactam as EXTENDED or
CONTINOUS INFUSION is suggested
(eg, 9g (3g of sulbactam) IV every 8H infused over 4H))
- narrowing to a single active agent to complete tx course can be considered after
sufficient clinical improvement is observed.
Polymyxins - can be considered in:
MILD: as monotherapy
MODERATE to SEVERE: combo with at least 1 other agent.
- colistin is suggested rather than polymyxin B for UTI CRAB.
- Both polymyxins has reliable in vitro activity against CRAB à polyB preferred
when considering polymyxin-based regimens, based on its more favourable
pharmacokinetic profile.
- Colistin is favoured for UTI à because it converts to its active form in urinary
tract.
** No CLSI susceptibility category for polyB against ACBA à most evidence
suggest that benefit with polymyxins would be diminished for poly
MICs>2mcg/mL.
- Rationale for combo tx in moderate to severe infection were due to 4 issues with
polymyxins:
 Concentrations of polymyxins in serum achieved with conventional
dosing strategies are highly variable & may be inadequate for
effective BACTERICIDAL activity.
 Dosage required to treat systemic infection approached the threshold
for nephrotoxicity à narrow therapeutic window.
 Activity of IV polymyxins in pulmonary epithelial lining fluid is
suboptimal à does not result in adequate bacterial killing in lungs.
 Several reports of CLINICAL FAILURE & resistance emergence
during polymyxin monotherapy.
Tetracycline - can be considered in:
MILD: as monotherapy
MODERATE to SEVERE: combo with at least 1 other agent.
- preferred à minocycline because of long standing clinical experience & availability
of CSI susceptibility interpretive criteria.
 - Alternative: High-dose tigecycline.
- tetracycline derivatives are capable of escaping common tetracycline resistant
mechanisms.
- emergence of resistance occurs thru efflux pump due to overexpression of various
RND-type transporters.
* Concern of usage à achieve rapid tissue distribution following administration à
limited concentrations in urine & poor serum concentrations.
- Dosage of minocycline: 200mg BD either IV or orally à no clinical failure signals
with its use & CLSI criteria available.
- Tigecycline:
 A derivative available as IV formulation
 No CLSI or FDA approved susceptibility criteria against CRAB
(minocycline cannot be used to predict susceptibility).
 Panel recommends high dose to be used.
- When used either tigecycline or minocycline as combo txà transitioning either
agent to a single active agent can be considered after appropriate clinical response, to
limit antibiotic-associated adverse events.
Meropenem - can considered as a component of combo tx in moderate to severe infections.
(extended-infusion)
- In vitro data suggests triple-combo tx consisting of:
(1) meropenem, unasyn, minocycline
(2) meropenem, unasyn, polymyxin B
à lead to bacterial eradication
- imipenem-cilastatin can be used as alternative & may retain activity against some
mero-R isolates.
- unaysn, meropenem (or imipenem-cilastatin) can be administered at high doses à
need to monitor because of the potential for additive BL toxicity, particularly
neurologic adverse events.
Cefiderocol - limited to treatment of CRAB refractory to other abx or in cases where intolerance
to other agents precludes their use.
- panel suggests agent should be prescribed as part of combo therapy.
- panel also suggests limiting consideration of cefiderocol for moderate to severe
CRAB infections after other regimens have been exhausted.
Rifamycins - includes agents: rifampicin, rifabutin, rifapentine à inhibit bacterial RNA
polymerase
- panel does not favour the use
Nebulized - does not suggest adding neb abx.
antibiotics for tx of
- in PK-PD modelling studies: aerosolised delivery of prodrug colistin to critically ill
respiratory
patients achieved high active drug level in epithelial lining fluid of lungs.
infections
- However, it is likely that neb abx do not achieve sufficient penetration &/or
distribution throughout lung tissue to exert significant bactericidal activity àdosage
 designed for IV formulations not inhalation.
- panel recommends AGAINST the use of neb abx as adjunctive therapy.

CLSI approved colistin AST test

 Q9: Pseudomonas DTR
Definition:
DTR is defined as P. aeruginosa exhibiting non-susceptibility to all of the following: piperacillin-
tazobactam, ceftazidime, cefepime, aztreonam, meropenem, imipenem-cilastatin, ciprofloxacin, and
levofloxacin.

Mechanism of resistant:
Decreased expression of outer membrane porins (OprD), hyperproduction of AmpC enzymes,
upregulation of efflux pumps, and mutations in PBP targets.

Treatment (IDSA):
Preferred antibiotics
Antibiotics Rationale/comments
Traditional non - tazosin, cefepime, ceftazidime, aztreonam.
carbapenem BL drugs - if tested SUSCEPTIBLE, they are preferred over carbapenem.
Carbapenem RESISTANT, - traditional agent as high-dose extended-infusion therapy is suggested, after
but susceptible to antibiotic susceptibility testing results are confirmed.
traditional BL (20-60%)
- MODERATE to SEVERE & poor source control: novel β-lactam agent that
tests susceptible (e.g., ceftolozane-tazobactam, ceftazidime-avibactam,
imipenem-cilastatin-relebactam).

- phenotype is generally due to lack of or limited production of OprD, which

normally facilitates entry of carbapenem agents into bacteria.

* When confronted with this kind of AST result, it is better to confirm

susceptibility.
If repeated testing shows same result, suggested tx à administer traditional BL
as high-dose extended infusion.

** Regardless of abx therapy, patients should be closely monitored to ensure clinical improvement as
P. aeruginosa exhibits an impressive capacity to acquire additional resistance mechanisms while
exposed to antibiotic therapy.
Disease Antibiotic Recommendation Rationale
Uncomplicated cystitis Preferred: - data shows non-inferiority of these agents
- Ceftolozane-tazobactam, ceftazidime-
avibactam, imipenem-cilastatin
relebactam, cefiderocol,

- or a single-dose of an aminoglycoside. - exclusively eliminated by the renal route

in their active form.
Alternative:
- colistin (not polyB)
- converts to its active form in the urinary
tract.

- oral Fosfomycin not recommended due to

high clinical failure (due to presence of
fosA gene is intrinsic to PSAE
Pyelonephritis & - Ceftolozane-tazobactam, ceftazidime-
uncomplicated cystitis avibactam, imipenem-cilastatin
relebactam, and cefiderocol.

- OD aminoglycoside - alternative option

Outside of urinary - Ceftolozane-tazobactam, ceftazidime- - administer as monotherapy.
tract avibactam, and imipenem-cilastatin - ceftolozane does not rely on inhibitor. It
relebactam. has independent activity.

Alternative:
- Cefiderocol

- Likelihood of the emergence of resistance of DTR PSAE during tx is highest for ceftolozane-
tazobactam and avikast.
- Emergence of ceftolozane-tazobactam resistance most commonly occurs because of amino acid
substitutions, insertions, or deletions in Pseudomonas-derived cephalosporinase (PDC), the
chromosomally encoded class C βlactamase of P. aeruginosa, commonly referred to as “the
pseudomonal AmpC”.
- Acquired resistance of P. aeruginosa to ceftazidime- avibactam is most frequently the result of
alterations in PDCs.
- Panel recommends always repeating AST for the newer β-lactams when a patient previously
infected with a DTR-P. aeruginosa presents with a sepsis like picture suggestive of a new or relapsed
infection.
Role of antibiotics
Antibiotic Role
Combination of antibiotics - Not routinely recommended if in vitro susceptibility to a first-line
antibiotic (i.e., ceftolozane tazobactam, ceftazidime-avibactam, or
imipenem-cilastatin-relebactam) has been confirmed.
- no additional benefit of combo therapy over monotherapy with BL.

- If no preferred agent - aminoglycoside (if susceptibility is demonstrated) can be

demonstrates susceptibility considered in combination with either ceftolozane-tazobactam,
ceftazidime-avibactam, or imipenem-cilastatin-relebactam,
preferentially selecting the BL-BLI agent for which the MIC is
closest to susceptibility breakpoint.

- If no aminoglycosides - polymyxin B can be considered in combination with the β-lactam-

demonstrate in vitro activity β-lactamase inhibitor.
- Polymyxin B is preferred over colistin for non-UTI because it is not
administered as a prodrug and therefore can achieve more reliable
plasma concentrations than colistin, and it has a reduced risk of
nephrotoxicity.

Nebulized antibiotics - does not recommend addition of neb abx as adjunctive therapy due
to the lack of benefit observed in clinical trials, concerns regarding
unequal distribution in infected lungs, and concerns for respiratory
complications such as bronchoconstriction.
 Q13: Stenotrophomonas maltophilia

- STMA is an aerobic, glucose NLF, GNB that is ubiquitous in the environment.

- produces biofilm & virulence factor that can enable colonization or infection in vulnerable hosts,
such as those with underlying lung disease & hematological malignancies (eg hemorhagic pn.)
- challenges in its management:
 Unclear whether STMA represents coloniser or true pathogen, particularly in pts with
UL pulmonary conditions such as CF or ventilator dependency.
 Often recovered as polymicrobial infectionà targeted STMA therapy is challenging!
 Presence of antimicrobial resistant genes further hampers treatment selection.
o L1 metallo-Blasé hydrolyses penicillins, cephalosporins, carbapenems, but
not aztreonam.
o L2 serine Blasé has extended cephalosporin activity & ability to hydrolyse
aztreonam.
o Both L1 & L2 render most conventional BL ineffective.
o Exhibits intrinsic resistance to AMINOGLYCOSIDES via chromosomal
aminoglycoside acetyl transferase enzymes.
o Accumulate multidrug efflux pumps that reduce activity of tetracyclines &
fluoroquinolones.
o Chromosomal Smqnr genes further reduces the effectiveness of
fluoroquinolone.
 ‘Standard of care’ abx regimen for STMA infections against which to estimate
effectiveness of various treatment regimens is not evident.
 STMA AST is problematic.
o Confidence in MIC interpretive criteria is undermined by concerns about
reproducibility of results for agents such as ceftazidime and levofloxacin
MIC testing methods that are commonly used in clinical laboratories, the
limited PK-PD data used to inform breakpoints for most agents, and
insufficient data to identify correlations between MICs and clinical outcomes.
o no CLSI susceptibility criteria established for the polymyxins.
o Incomplete STMA growth inhibition often occurs in polymyxin wells,
suggestive of heteroresistance

General approach for the treatment

Disease Recommendations
severity
MILD - Bactrim, minocycline, tigecycline, levofloxacin or cefiderocol monotherapy.
(& - preferred: Bactrim & minocycline
polymicrobial - NOT SUGGESTED: ceftazidime
) (Why? àdue to intrinsic BLasé produced likely to render CEF ineffective)
- * Levofloxacin should be administered with caution à potential for combination of efflux pumps &
Smqnr genes renders tx ineffective.
MODERATE - Any of the 3 approaches:
to SEVERE 1) combo therapy with Bactrim & minocycline (preferred)
2) initiation of bactrim monotherapy with addition of 2nd agent (minocycline (preferred), tigecycline,
levofloxacin or cefiderocol) if there is delay in clinical improvement with Bactrim alone.
 3) combo ceftazidime-avibactam & aztreonam, when intolerance or inactivity of other agents are
anticipated.
Role of antibiotics
Antibiotics Rationale/ Role
Bactrim - MILD: preferred monotherapy
- Combo if moderate to severe infection.
Tetracycline - MILD: High-dose minocycline monotherapy
- MODERATE to SEVERE: High-dose minocycline in combo with 2nd active agent, at least
until clinical improvement is observed.
- minocycline & tigecycline have good lung tissue penetration.
- Not suggested for STMA UTI à achieve rapid tissue distribution following administration
- endorsed only as component of combo therapy for tx of BSI, at least until clearance of
blood cultures & after clinical improvement is observed.
Fluoroquinolone - Levofloxacin monotherapy à tx option for mild infections
s - MODERATE to SEVERE: combo with second active agent (Bactrim, minocycline,
tigecycline or cefiderocol).
- Several studies have shown that STMA isolates that test susceptible to levofloxacin can
develop elevated levofloxacin MICs during therapy.
- although can be considered as monotherapy for mild infections, panel suggests to only use
as component of combo therapy, preferably with Bactrim for tx moderate to severe infection.
- adverse events related to fluroquinolone use & potential for emergence of R STMA during
levofloxacin should be considered when prescribing this agent.
Cefiderocol - may be adequate as monotherapy for mild infections.
- combo with second agent for treatment of mod-severe infection, at least til clinical
improvement is observed.
Ceftazidime- - combo of both drugs is suggested for moderate to severe infection, when neither Bactrim or
avibactam & minocycline are considered viable tx options.
aztreonam - combo can be used to overcome activity both both the L1 and L2 β-lactamases.
- L1 metallo-BL hydrolyzes ceftazidime-avibactam but not aztreonam.
- L2 serine BL inactivates ceftazidime and aztreonam but is inactivated by avibactam.
- reasonable tx, such as in BSI or PN in hematologic malignancy population & where
intolerance or resistance to other agents precludes their use.
Ceftazidime - does not suggest prescribing ceftazidime for the treatment of STMA infections, as intrinsic
L1 and L2 β-lactamases are expected to render it ineffective.
- Almost 30- 40% of isolates test susceptible to ceftazidime using CLSI interpretive criteria
- Local labs and antibiotic stewardship teams are encouraged to convey the likely
ineffectiveness of ceftazidime against STMA, even when it tests susceptible, to clinicians.
- An additional concern potentially related to the presence of inactivating β-lactamases is that
ceftazidime MICs against STMA may be inaccurate and non-reproducible using
susceptibility methods commonly employed by labs.
Q12: Inducible Clindamycin resistant testing

- erythromycin (macrolides) & clindamycin (licosanamides) have similar mechanism of action

(inhibition protein synthesis) & mechanism of resistance.
- 2 main mechanism of macrolide resistant:
o Efflux pump that only affects macrolides
 Confers resistance to macrolides only (M-type resistance, for
macrolide only)
 Mediated in Staphylococcal by msrA.
o Methylase that alters binding site for both abx (erythromycin & clindamycin)
 Confers resistance to macrolides, lincosanamide & streptogammin B
(MLSB-type)
 Mediated by an erm gene (usually ermA or ermC).

- In Staphylococci, MLSB-type can be either constitutive or inducible

o If it is inducible, isolate appears to be susceptible to clindamycin (liconsamide),
unless it is induced by macrolide (erythromycin).
o Important to determine whether it is constitutive or inducible, when it is considered
for therapy.
- If isolate has M-type resistance, it is resistance to erythromycin but susceptible to clindamycin.
- If isolate has MLSB- type resistance, it is resistant to erythromycin, but can test susceptible to
resistant to clindamycin, depending on whether it is constitutive/inducible.
- * for strains that are erythromycin resistant but clindamycin intermediate or susceptible à
determine whether inducible clindamycin resistant exits (due to erm gene) or if remains clindamycin
susceptible (due to efflux gene).
- Same logic goes to Streptococci, can occur in beta-Strep, STPN & viridians group.
- M phenotype in Strep is due to mefA & gene responsible for MLSB type is due to ermB (rarely
ermTR).

2 test methods:
- D-zone test (CLSI & EUCAST)
- broth dilution test (CLSI)

CLSI now recommends that isolates that are D-zone positive or grow in the combination well to be
reported as CLINDAMYCIN RESISTANT.

Suggested Comments to include in report:

CLSI: this isolate is presumed to be resistant based on detection of inducible clindamycin R.
EUCAST: potential use of clindamycin for short term therapy of less serious infection caused by
isolates with inducible clinda R, because emergence of resistant is not anticipated during tx.
1) D-zone test:
- use disc diffusion method.
- place 15ug erythromycin disc adjacent to 2ug clindamycin disc à look for flattening of clindamycin
zone (looks like D)
- if test is done for staphylococci à place disc 15 to 26mm apart (clsi)
à 12 to 20mm apart (EUCAST)

- For Streptococci, disc placed 12mm apart on MHA supplemented with 5% sheep BA (CLSI).
- Clindamycin alone is a poor inducer of the methylase
- if positive à indicates the presence of macrolide-inducible resistance to clindamycin produced by an
inducible methylase that alters the common ribosomal binding site for macrolides, clindamycin and
the group B streptogrammins.

2) single-well broth dilution method

- refer clinical microbiology manual pg 1327, volume 1. (Hopefully this method doesn’t come out in
the exam!!)
Breakthrough infection or persistent bacteremia (prof Zak’s class..?viva)
Causes:
1) Pathogen factor:
- virulence of pathogen, for example STAU or Candida has propensity to form biofilm on abiotic
surfaces which can propagate, detach and seed in other organs.
- formation of biofilm allows persister cells to survive, intrinsic resistant to host immune system,
endotoxin production and provides a niche/reservoir for resistant bacteria.
- in a situation where there is persistent bacteremia, despite appropriate source control and apparently
adequate antibiotic after 2 to 3 days, we need to exclude metastatic foci of infection.
Examples of metastatic foci of infection: infective endocarditis, septic arthritis, osteomyelitis,
abscesses.

2) Host factor
- changes in the host that facilitate establishment of persistent infection.
For example: presence of foreign device (eg CVC, prosthetic joint, prosthetic heart valves)
- Device associated infections represent one of the most common forms of persistent infections.
- It is estimated that more than 50% of hospital-acquired infections are due to infected medical
devices, including prosthetic joints, central venous catheters (CVCs), and prosthetic heart valves.
- Underlying co morbids affecting host immune defense
For example: defective innate immune system underlies diabetics’ susceptibility to bacterial infections
à impaired phagocytic function, such as chemotaxis, phagocytosis, respiratory burst.
- reduced antibiotic dosage (compared to standard dose) in patients with underlying renal dysfunction
(? Not sure if consider a point, pts with renal or hepatic dysfunction may alter the metabolism and
elimination of antibiotics, leading to increased concentrations and potential toxicity).
- Augmented renal clearance à define as urinary creatinine clearance more than 130
mL/min/1.73m2.
o present in 20-65% of critically ill patients
o risk factors for ARC are male sex, young age, trauma admission, and brain injury
(traumatic and subarachnoid haemorrhage).
o Sepsis alters PK properties of Plasma concentrations of hydrophilic antibiotics (β-
lactams, glycopeptides and aminoglycosides). They have an increased volume of
distribution (Vd) leading to a lower-than-expected maximum serum concentration
during a dosing interval (Cmax)
o Additionally, those antibiotics predominantly eliminated by the kidney, may be
significantly decreased in patients with high values of CLCR
- Renal replacement therapy (RRT) and extracorporeal membrane oxygenation (ECMO) will also
affect the PK of antibiotics

3) Antibiotic factor
- Specific regions in host where physical structures may obstruct affective immune response &
prevent adequate penetration of antibiotic, notably BBB, joint spaces & sinuses.
- Route of administration: extended infusion vs bolus of meropenem in CRE treatment.
- Dosage of antibiotic: Ex high dose unaysn vs standard dose in the treatment ACBA XDR infection.
- Administration of abx with inadequate PK/PD target attainment may potentiate the development of
antibiotic resistance.
 For ex: patients in critical care who are at high risk of resistant organism, received suboptimal
empirical antibiotic.

Synergic Testing
- Due to the increasing prevalence of drug-resistant organisms and limited
options for the treatment of these infections, testing for antimicrobial
interactions has gained popularity.
- In vitro combination testing provides information, on which two or more
antimicrobials can be combined for a good clinical outcome.
- Time-kill assay (TKA), checkerboard (CB) assay and E-test-based methods are
most commonly used.
- Comparative performance of these methods reveals the TKA as the most
promising method to detect synergistic combinations followed by CB assay and
E-test.
- Reasons for performing synergic testing:
(i)necessity to extend the antimicrobial spectrum,
(ii) possibility of reducing the dosage and toxicity
(iii) possibility of reducing the development of resistance
(iv) emergence of MDR/XDR organisms and the lack of development of new abx

Rationale behind testingà choice of combination therapy is that the

antimicrobials will have a synergistic effect when given together.

Assay Principle Advantages Disadvantages

Time-kill - determines actual reduction in the viable count of the - standard - limited abx
assay (TKA) organism after exposure to the drug combination reference assay concentrations,
compared to the most active single agent at different time non-
intervals. standardised
- add a standard inoculum in broths containing inoculum size
the individual abx and its combination and abx
- Sub-culturing is done from the broth concentration,
containing antimicrobials at different time static
intervals and the bacterial count is done . concentration of
- colony count is done at different time intervals the drug
depending on whether abx exhibits time-dependant killing
or concentration-dependant killing. - labour
- interpretation: intensive and
Synergic action: ≥2 log10 CFU/ml reduction in time-consuming
the bacterial growth in the combination when
compared to the most active single agent.
Antagonist: ≥2 log10 CFU/ml in the combination
compared to the most active single agent.
Bactericidal effects
of the combinations are determined by a
decrease of ≥3 log10 CFU/ml from the initial
inoculum.
Checkerboar - utilises a panel of antimicrobial - Relatively - Different
d assay combinations at different concentrations easy methods for
either in the - Multiple interpretation
macrobroth (2 ml volume) or microbroth (100 concentrati
- Intra-assay
µl volume) ons tested variation
method.
- range of tested concentrations varies from 4 - Static
to 8 times the MIC to at least 1/8–1/16 of the concentration of
MIC. drugs
- Interpretation:
Fractional inhibitory concentration (FIC) is
calculated for each antibiotic at a given
concentration combination by the following
formula:
FIC of agent A = MIC of agent A in
combination/MIC of agent A alone.
- Cumulative FIC is then calculated by
summing up the FIC
of both the agents.
- ‘Synergy’ is interpreted when the FIC index
is ≤0.5,
- ‘indifference’ or ‘no interaction’
corresponds to the FIC index >0.5–4.0
- ‘antagonism’ when the FIC index is >4.0
E test 1) Cross method - Easy to - detects less
methods - MHA plate is inoculated with 0.5 perform synergy
McF matched inoculum, to which E-test strips - different
are placed 1 over the other at 90° angle methods
crossing at the MICs of the - restricted to 2
individual agent of the organism determined drug combos
earlier. only.
- After incubation for 18 h, the zone of
inhibition is read and FIC is determined based
on formula above.
2) Fixed ratio method
- MHA plates are inoculated with 0.5 McF matched
inoculum.
- E-test strip of the 1st agent is placed and
incubated at room temperature for 1 h to allow the abx to
diffuse into the medium.
- After 1 h, it is removed and saved
as MIC template.
- E-test strip for the 2nd agent is then
placed directly over the imprint of the first strip.
- FIC is then determined.

3) Agar test method

- MHA plates are incorporated with 0.5 × or
0.125 × MIC of one agent and the E-test strip
of the second agent is placed over the
inoculated surface.
- MIC obtained is compared with the MIC in
drug-free medium.
- synergy is interpreted when there is more
than 3-fold reduction in MIC on the drug-
incorporated medium.

4) MIC:MIC method
- one test strip is placed on the inoculated
MHA plate and incubated at room T for 1 h to
allow diffusion of the agent.
- After 1 h, the agar is marked adjacent
to the previously determined MIC of the agent
 and removed.
- second E-test strip is then placed over the
imprint of the previous strip such that the
mark on the agar corresponds to
MIC of the second agent.
- Resulting ellipse of inhibition is read after 20
h of incubation at 37°C.
- FIC index is calculated and interpreted.

** other synergy tests to refer journal