LABORATORY DETECTION OF RESISTANT BACTERIA

Dr Mostafa Mahmoud, MD, PHDConsultant Microbiologist

Associate Prof of Microbiology & Immunology

History of antimicrobials & resistance

• Bacteria are said to have been present 3,500 million years ago, causing severe morbidity and high rates of mortality before the discovery of antimicrobial agents. Antimicrobial agents were known and used for the control of bacterial infections in the form of mercury, bismuth and other heavy metals in the early 1400s, when, for example, mercury was used in the treatment of syphilis caused by Treponemapallidum.

• However, These agents were toxic to the host.

• In 1908, Paul Ehrlich and colleagues discovered Salvarsan, which is an arsenic compound used in the treatment of syphilis. Ehrlich was offered the Nobel Prize for this “magic bullet”, as he termed it (Harmful Side effects).

• The first conventional antimicrobial drug discovered was sulfonamide by Bayer in 1932.

• This was followed in the early 1940s by the use of penicillin compounds by Florey and his colleagues in Oxford, based upon the discovery of Alexander Fleming’s natural penicillin.

• streptomycin in 1944, chloramphenicol in 1947and chlortetracycline in 1948

• In contrast to the common belief which considers antimicrobial resistance to be a new phenomenon, it is actually older.

• in 1907 during the work by Ehrlich in trying to discover an agent for the drug-resistant Trypanosoma brucei, which was known as “drug-fast”.

• Bacterial antimicrobial drug resistance was increasing and becoming more complicated with the introduction of further antimicrobial drugs in the 1950s, and was developed for many antimicrobials at the same time by single microorganisms which are known as multidrug-resistant (MDR) bacteria.

Effects of antimicrobial resistance:

1- increase in the rate of the morbidity and mortality related to microbial infections.

2- increase in expenses for treatment and research.

3- increase in the number of hospital stays.

4- delay in some operative interventions.

5- more costs due to isolation procedures.

Cellular targets for some antimicrobials

Mechanisms of antimicrobial drug resistance:

A- Natural resistance

• An organism is termed as having natural resistance when it is resistant to the action of an antibiotic from the start; this pattern of resistance is common to all isolates of the species, e.g. the resistance of Escherichia coli to macrolides.

• It is explained by the absence or inaccessibility of the target for the drug action, e.g. Amphotericin B does not act upon bacteria due to the absence of sterols and Gram-negative bacteria are naturally resistant due to the non-permeability of the outer membrane.

• Acquired resistance

• It is developed to an antibiotic to which the microorganism was previously susceptible; it develops with one or more isolates of the species, i.e. not all strains of a species are resistant.

• The genetic mechanisms of acquired resistance are either due to arise of a mutation or transferof resistant genes by conjugation, transduction, transformation or transposition.

Different gene transfer mechanisms in bacteria: A =transformation, B = transduction, and C= conjugation

The molecular mechanisms of resistance:

SN Mechanism Examples of affected

antimicrobials

1 Destruction, modification, or

inactivation of the antimicrobial.

β-lactam antibiotics

Chloramphenicol

Aminoglycosides

2 Multidrug efflux pumps. Tetracycline

3 Target site alteration. β-lactam antibiotics

Chloramphenicol streptomycin

Quinolones

Fusidic acid

Erythromycin

Glycopeptides

Rifampicin

4 Reduction in the cell surface

permeability or access of the

antimicrobial to the cell interior.

Tetracyclines

Quinolones

β-lactam antibiotics

Aminoglycosides

Chloraphenicol

5 New metabolic bypass mechanism. Rrimethoprim

Sulphonamides

mechanisms of antimicrobial resistance in Gram-negative bacteria

MDRO: Definition

• Multidrug-Resistant Organisms (MDROs) are

defined as microorganisms that are resistant to one

or more classes of antimicrobial agents.

• Three most common MDROs are:

1. Methicillin-Resistant Staph aureus (MRSA)

2. Vancomycin Resistant Enterococci: (VRE)

3. Extended Spectrum Beta-Lactamase producing

Enterobacteriaceae. (ESBLs)

1- Detection of Extended-Spectrum β-Lactamases (ESBLs) production.

• What are the ESBLs?

• ESBL is an enzyme (constitutive, acts just when

expressed) produced by certain enterobacteria (E.

coli, Klebsiella & Proteus) enabling them to resist

(hydrolyze) all penicillins, aztreonam,

cephalosporins (including cefipime) but not

cephamycins like (cefoxitin, Cefotetan) or

Carbapenems.

Why should clinical laboratory personnel be concerned about detecting

these enzymes?

• Difficult to detect.

• Various levels of resistance to Beta Lactams.

CLSI 2010 ESBLs testing for Klebs. pneumoniae, K. oxytoca, or E. coli

Disc diffusion Broth dilution

Antimicrobial Content Resistant Zone Resistant MIC

Init

ial t

esti

ng Cefpodoxime 10 μg ≤ 17 mm ≥ 8 μg/mL

Ceftazidime 30 μg ≤ 22 mm ≥ 2 μg/mL

Aztreonam 30 μg ≤ 27 mm ≥ 2 μg/mL

Cefotaxime 30 μg ≤ 27 mm ≥ 2 μg/mL

Ceftriaxone 30 μg ≤ 25 mm ≥ 2 μg/mL

Co

nfi

rmat

ory

Tes

tin

g

CeftazidimeCeftazidime -clavulanic acid

30 μg30/10 μg

A ≥ 5-mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid vs its zone when tested alone = ESBL (e.g., ceftazidimezone = 16; ceftazidime/clavulanicacid zone = 21).

A ≥ 3 twofold concentration decrease in an MIC for either antimicrobial agent tested in combination with clavulanicacid (4 μg) vs its MIC when tested alone = ESBL (e.g., ceftazidime MIC = 8 μg/mL; ceftazidime-clavulanic acid MIC = 1 μg/mL).

CefotaximeCefotaxime -clavulanic acid

30 μg30/10 μg

CLSI recommendations for ESBLs• CLSI recommends performing phenotypic confirmation

of potential ESBL-producing isolates of K. pneumoniae, K. oxytoca, or E. coli by testing both cefotaxime and ceftazidime, alone and in combination with clavulanicacid. Testing can be performed by the broth microdilution method or by disk diffusion.

• - For MIC testing, a decrease of > 3 doubling dilutions in an MIC for either cefotaxime or ceftazidime tested in combination with 4 µg/ml clavulanic acid added to all dilutions, versus its MIC when tested alone, confirms an ESBL-producing organism e.g. MIC to CAZ = 8; to CAZ/CLAV = 1 (8,4,2,1).

• - For disk diffusion testing, a > 5 mm increasein a zone diameter for either antimicrobial agent tested in combination with clavulanicacid (10 µg) versus its zone when tested alone confirms an ESBL-producing organism.

• Note there is different figures for Proteus mirabilis (see original text).

• For confirmed ESßL-producing bacteria, report should express all the following as resistant even if they are susceptible in vitro:

• – All penicillin’s

• – All cephalosporins

• - Aztreonam

• For confirmed ESßL-producing bacteria, report should not override (no change in susceptibility interpretations) for:

• – Cephamycins (e.g. cefoxitin).

• - Beta-lactam/beta-lactamase inhibitor combinations (e.g. piperacillin/ tazobactam)

• – Carbapenems

Phenotypic detection of ESBL

Detection of ESBL by E-test

2- Detection of resistance to Carbapenems

• Carbapenems are Bactericidal, beta lactamfamily of antibiotics, target cell wall, disrupt different stages in peptidoglycan synthesis.

• The FDA approved for clinical use:

imipenem, meropenem, ertapenem, and doripenem

• They are broad-spectrum.

• Used in treatment of life-threatening infections:

- Septicemia

- MDR GNB

Causes of Carbapenem Resistance:

1- Impaired permeability due to porinmutation.

2- Efflux pumps.

3- “Carbapenemase-hydrolyzing enzymes”

• There are 4 classes of Carbapenemases; A, B, C, and D.

• Class C is very rare clinically.

Cabapenemase Ambler classification system

Class Enzyme Most common

bacteria

Inhibitor

Class A Chromosomal:

IMI, SME, NMC

Plasmid:

KPC, GES

Enterobacteriaceae Boronic Acid

(clavulanic)

Class B Metallo-β-lactamases:

IMP, GIM, VIM, SPM,

NDM

Ps. aeruginosa,

Enterobacteriaceae

Acinetobacter

EDTA

Class D OXA β-lactamases:

Oxa-23, Oxa-48,

Ps. Aeruginosa,

Enterobacteriaceae

Acinetobacter

Oxacillin

Laboratory Approach to Carbapenemases Identification

1. Disc diffusion with meropenem 10 μg on Mueller Hinton agar (MHA) or broth dilution.

2. Phenotypic confirmation

– Modified Hodge test

– Microbiological tests with inhibitors

3. Genotypic confirmation

– Molecular tests for detection of the related gene e.g. PCR.

Clinical Breakpoints for Carbapenemases detection (CLSI 2010 recommendations):

Antibiotic MIC 2010 MIC Before

2010

Disc diffusion

(10 μg)

S I R S R S I R

Imipenem < 1 2 > 4 < 4 > 16 > 23 20-22 < 19

Meropenem < 1 2 > 4 < 4 > 16 > 23 20-22 < 19

Ertapenem < 0.25 0.5 > 2 < 2 > 8 > 22 20-22 < 18

Doripenem < 1 2 > 4 < 4 ND > 23 20-22 < 19

Phenotypic confirmation Modified Hodge Plate

• The organism in the background of the plate is carbapenemsusceptible strain (E.coli ATCC 25922) on MHA.

• Drawbacks: Not identify the class – false negative

Hodge test for many strains:

Usage of carbapenemase inhibitors:1-Boronic Acid Synergy (combination)

• Boronic acid inhibits group A Carbapenemases.

• The same test format can be done with combination discs of EDTA, Oxacillin, for other classes of carbapenemases.

• Commercial kits are available for class differentiation (Rosco kit).

• Proteus spp., Providencia spp., and Morganellaspp. may have elevated MICs to imipenem by mechanisms other than production of carbapenemases; thus, the usefulness of the imipenem MIC screen test for the detection of carbapenemases in these three genera is not established.

Other laboratory tests:1– Confirmation of Meropenem resistance by E-test,

2– Colistin, Tigecycline E-tests

• – No CLSI interpretative criteria

3. Genotypic confirmation (Gold Standard)

• PCR assays can target different genes in single or multiplex formats.

– Class A (KPC, IMI, NMC, SME),

– Class B (IMP, VIM, AIM, GIM, KHM, SIM, SPM, and NDM)

– Class D (OXA-23-like, OXA-24-like, OXA-48-like, OXA-51-like and OXA-58-like)

3- MRSA Screening• The MRSA are either healthcare associated (HA-

MRSA) or community acquired (CA-MRSA).

• It is resistant to anti-beta lactamase penicillin e.g. oxicillin, methicillin. Cloxacillin, flucloxacillin,

• The mechanism of resistance is via chromosomally mediated mecA gene which mediates the expression of a different penicillin Binding protein called (PBP-2a) which does not bind these drugs making the organism resist to them.

• MRSA screening depend upon PCI policy.

• Criteria for MRSA:

1- The presence of the mecA gene.

2- Either an oxacillin MIC of > 2 μg/mL,

a methicillin MIC of > 4 μg/mL, or

a cefoxitin MIC of > 4 μg/mL.

(The most reliable one is cefoxitin).

Detection methods are:1- Conventional by culture on:

– Mannitol slat agar (MSA) with 7% NaCl or chromogenic agar with high salt content.

– Enrichment on nutrient broth with high salt content then subculture on MSA.

2- Molecular method: by detection of the mecAgene by PCR.

• MSA is not suitable for running serological testing for identification of Staph aureus also slow growth.

Mannitol Salt agar

MRSA chromogenic agar (Hardy)

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

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

CLSI Screening Tests for β-LactamaseProduction, Oxacillin Resistance, and mecA-Mediated Oxacillin Resistance Using Cefoxitinin the S. aureus.Screen test β-Lactamase Oxacillin Resistance mecA-Mediated Oxacillin Resistance Using

Cefoxitin

Organisms S. aureus with penicillinMICs ≤ 0.12 μg/mL or zones ≥ 29 mm

S. aureus S. aureus and S. lugdunensis

Test method Disk diffusion Nitrocefin-based test Agar dilution Disk diffusion Broth microdilution

Medium MHA NA MHA with 4%NaCl MHA CAMHB

AMConc.

10 U disc penicillin NA 6 μg/mL oxacillin 30 μg Disc ofcefoxitin

4 μg/mL cefoxitin

Inoculum Standard disk diffusion

Induced growth1 Direct colony suspension2

Standard disk diffusion

Standard brothmicrodilution

Incubationconditions

35 ± 2°C; ambient air Room temperature 33–35°C; ambient air.(Testing at emperatures above 35°C may not detect MRSA.)

Incubationlength

16–18 hours Up to 1 hour 24 hours; 16–18 hours 16–20 hours

Results Sharp zone edge(“cliff”) =β-lact. positive.Fuzzy zone edge(“beach”) =β-Lact. negative.

Nitrocefin-based test:conversion from yellow tored/pink =β-lactamase positive.

Examine carefully with transmitted light for > 1colony or light film of growth.> 1 colony = oxacillinresistant.

≤ 21 mm = mecA positive≥ 22 mm = mecAnegative

> 4 μg/mL = mecApositive≤ 4 μg/mL = mecAnegative

Screen test β-Lactamase Oxacillin Resistance mecA-Mediated Oxacillin Resistance UsingCefoxitin

Furthertesting andreporting

β-Lactamase-positive staphylococci are resistant topenicillin, amino-, carboxy-, andureidopenicillins.

Oxacillin-resistant staphylococci are resistant toall β-lactam agents; other β-lactam agentsshould be reported as resistant or should not bereported.

Cefoxitin is used as a surrogate for mecA-mediatedoxacillin resistance.Isolates that test as mecA positive should be reported asoxacillin (not cefoxitin) resistant; other β-lactam agentsshould be reported as resistant or should not be reported.Because of the rare occurrence of oxacillin resistancemechanisms other than mecA, isolates that test as mecAnegative, but for which the oxacillinMICs are resistant (MIC≥ 4 μg/mL), should be reported asoxacillin resistant.

QCrecommend-ations

S. aureus ATCC® 25923S. aureus ATCC®29213

S. aureus ATCC® 29213 positiveS. aureus ATCC® 25923 negative

S. aureus ATCC® 29213 – Suscep.ATCC® 43300 –Resistant

S. aureus ATCC® 25923 mecA negative (zone 23–29mm)S. aureus ATCC® 43300 mecA positive (zone ≤ 21 mm)

S. aureus ATCC® 29213 mecA negative (MIC 1–4 μg/mL)S. aureus ATCC® 43300 mecA positive (MIC> 4 μg/mL)

4- Vancomycin-Intermediate/Resistant Staphylococcus Aureus (VISA/VRSA)

laboratory detection

• Most SA are VSSA with MIC of 0.5-2 μg/mL.

• Vancomycin MIC of VISA is 4-8 μg/mL.

• Vancomycin MIC of VRSA is ≥ 16 μg/mL.

• No CLSI disc diffusion criteria for VISA or VRSA(only for VSSA >=15 mm).

Laboratory detection of VRSA• VRSA are detected by:

- Reference broth microdilution,

- agar dilution,

- E-test®,

- MicroScan® overnight and Synergies plus™;

- BD Phoenix™ system,

- Vitek 2™ system, disk diffusion, and

- The vancomycin screen agar plate [brain heart infusion (BHI) agar containing 6 µg/ml of vancomycin].

What are the mechanisms of resistance for VRSA and VISA?

• All VRSA isolates to date contained the vanAvancomycin resistance gene.

• The vanA gene is usually found in enterococciand typically confers high-level vancomycinresistance (MICs= 512-1024 µg/ml) to these organisms (VRE).

• There is also VanB and VanC genes.

Should VISA and VRSA be reported to the infection control team?

• Yes and you have to notify also the lab

admin about any suspicious isolates.

Thank You