**3. Phenotypic tests**

Phenotypic tests may be used in the everyday laboratory practice in order to identify the presence of acquired resistance mechanisms among frequently isolated nosocomial pathogens. In the present chapter, the procedure and the interpretation of seven useful phenotypic tests are described. Special attention has been given to the phenotypic detection of beta-lactamases and especially those hydrolyzing carbapenems together with other beta-lactams (carbapene‐ mases).

## **3.1. Double Disc Synergy Test (DDST)**

The DDST is used for the detection of beta-lactamases that are inhibited by beta-lactamase inhibitors such as clavulanic acid (Ambler class A beta-lactamases and especially ESBLs). For SPICE organisms, cloxacillin should be incorporated in Mueller-Hinton agar during its preparation in order to prevent any AmpC interference [27].

#### *3.1.1. Procedure*

Four general mechanisms leading to acquired antibiotic resistance have been described: (1) decreased entrance of the antibiotic into the bacterial cell; (2) increased extrusion of the antibiotic by bacterial efflux systems; (3) mutational modification of the antibiotic's target and; (4) production of antibiotic-inactivating enzymes. Characteristic examples for each mechanism

**Decreased permeability** Diminished expression or loss of the OprD porin in *Pseudomonas aeruginosa* and

OqxAB in *Klebsiella pneumoniae* [10-13] **Target modification** Mutations of gyrases and topoisomerases leading to fluoroquinolone resistance

**Inactivating enzymes** Production of beta-lactamases and aminoglycoside modifying enzymes [17-19]

Among the aforementioned mechanisms, the production of beta-lactamases is considered of major importance because these enzymes are commonly transferable and inactivate multiple beta-lactam antibiotics. Within this large enzymatic family, carbapenemases (class B metallobeta-lactamases (MBLs) [20] that contain zinc in their active center and class A KPC [21]) hydrolyze in vitro all or almost all beta-lactams, including carbapenems [22]. Class A extended spectrum beta-lactamases (ESBLs) hydrolyze penicillins, monobactams and cephalosporins whereas are inhibited by the beta-lactamase inhibitors [23,24]. Class C cephalosporinases (AmpC) present various spectrums of cephalosporin hydrolysis but are not inhibited by the beta-lactamase inhibitors [25]. Additionaly, AmpC enzymes may be inducible in *Serratia* spp, *Pseudomonas* spp, Indole-positive *Proteus*, *Citrobacter* spp and *Enterobacter* spp (SPICE group of bacteria) complicating the treatment of infections caused by these pathogens. Finally, molecular class D beta-lactamases (OXA) comprise numerous enzymes with variable spec‐

Phenotypic tests may be used in the everyday laboratory practice in order to identify the presence of acquired resistance mechanisms among frequently isolated nosocomial pathogens. In the present chapter, the procedure and the interpretation of seven useful phenotypic tests are described. Special attention has been given to the phenotypic detection of beta-lactamases and especially those hydrolyzing carbapenems together with other beta-lactams (carbapene‐

OmpK35, OmpK36 porins in *Klebsiella pneumoniae* [6-9] **Efflux** Overexpression of MexAB-OprM and MexXY-OprM in *Pseudomonas aeruginosa* and

**Mechanism Examples**

[14-16]

**Table 1.** Examples of antibiotic resistance mechanisms.

trums of beta-lactam hydrolysis [26].

**3. Phenotypic tests**

mases).

are presented in Table 1.

140 Trends in Infectious Diseases

**Figure 1.** Double Disc Synergy Test preparation.

Step 1: Prepare agar plates containing 200μg/ml cloxacillin (by adding 1ml solution containing 80 mg cloxacillin in 399 ml Mueller-Hinton agar at the liquid phase). Omit this step when testing non-SPICE bacteria.

Step 2: Make a 0.5 McFarland bacterial suspension.

Step 3: Inoculate with a sterile cotton swab and place an amoxicillin/clavulanic acid disc at the center of the plate (20 μg amoxicillin+10 μg clavulanic acid).

Step 4: Place ceftazidime, imipenem, ceftriaxone, cefotaxime, aztreonam and cefepime discs around the central amoxicillin/clavulanic acid disc (Figure 1).

Step 5: Incubate at 37o C for 18-24h.

#### *3.1.2. Interpretation*

The DDST is considered positive when the inhibition zone of any of the antibiotics is larger towards the clavulanic acid disc (Figure 2-lower left plate) or a ghost inhibition zone appears between the central disc and any of the other antibiotics (Figure 2-lower right plate). This is happening because of the ESBL's inhibition by the clavulanic acid. In proximity to the central disc the enzyme's activity is blocked. Thus, the growth inhibition zone appears only towards the clavulanic acid disc. If resistance to cephalosporins is not due to ESBL production, the test results negative (Figure 2-upper plates).

*3.2.2. Interpretation*

**3.3. Boronic acid test**

*3.3.1. Procedure*

Step 4: Incubate at 37o

*3.3.2. Interpretation*

alone (Figure 6).

The imipenem-EDTA synergy test is positive when the inhibition zone takes a characteristic keyhole shape because of the MBL inactivation by the EDTA (Figure 4). In proximity to the EDTA discs, the hydrolytic activity of MBLs is blocked. Consequently, imipenem and cefta‐

Phenotypic and Molecular Methods for the Detection of Antibiotic Resistance Mechanisms in…

http://dx.doi.org/10.5772/57582

143

Phenylboronic acid acts as an inhibitor for KPC carbapenemases and class A and C betalactamases. The boronic acid test has been proposed for the phenotypic detection of KPCproducers because it is easier to perform than the DDST and also presents less false positive

Step 2: Inoculate with a sterile cotton swab and place two meropenem discs (Figure 5).

**Figure 4.** Interpretation of the imipenem-EDTA synergy test. (-): Negative; (+): Positive. Note that the upper left isolate is negative for MBL production but shows positive D-test between imipenem and ceftazidime indicating for the pres‐

In case of KPC production, the phenylboronic acid that has been added to the second mero‐ penem disc will block the hydrolytic activity of the enzyme. As a consequence, the second disc will have a larger inhibition halo. The test is considered positive when the inhibition zone of the meropenem+phenylboronic acid is ≥ 5mm larger than the inhibition zone of meropenem

Step 3: Add 20 μl of phenylboronic acid 20 g/L on one of the two meropenem discs.

ence of inducible AmpC beta-lactamases (The D-test is described in paragraph 3.6).

C for 18-24h.

zidime inhibition zones may appear larger towards the EDTA discs.

results because of the presence of ESBLs or AmpC beta-lactamases [29-31].

Step 1: Make a 0.5 McFarland bacterial suspension.

**Figure 2.** Interpretation of the DDST. (-): Negative; (+): Positive.

#### **3.2. Imipenem-EDTA synergy test**

EDTA (ethylene-diamine-tetraacetic acid) is a polyamino carboxylic acid that binds metal ions like zinc and can inactivate the metallo-beta-lactamases. Therefore, it is used for the phenotypic detection of MBL production in clinical isolates [28].

#### *3.2.1. Procedure*

Step 1: Soak paper discs within a 0.1 M EDTA solution.

Step 2: Make a 0.5 McFarland bacterial suspension.

Step 3: Inoculate with a sterile cotton swab and place an imipenem and a ceftazidime disc at the center of the plate.

Step 4: Place the EDTA discs at both sides in respect to the antibiotics as shown in Figure 3.

Step 5: Incubate at 37o C for 18-24h.

**Figure 3.** Preparation of the imipenem-EDTA synergy test.

### *3.2.2. Interpretation*

The imipenem-EDTA synergy test is positive when the inhibition zone takes a characteristic keyhole shape because of the MBL inactivation by the EDTA (Figure 4). In proximity to the EDTA discs, the hydrolytic activity of MBLs is blocked. Consequently, imipenem and cefta‐ zidime inhibition zones may appear larger towards the EDTA discs.

#### **3.3. Boronic acid test**

Phenylboronic acid acts as an inhibitor for KPC carbapenemases and class A and C betalactamases. The boronic acid test has been proposed for the phenotypic detection of KPCproducers because it is easier to perform than the DDST and also presents less false positive results because of the presence of ESBLs or AmpC beta-lactamases [29-31].

### *3.3.1. Procedure*

**Figure 2.** Interpretation of the DDST. (-): Negative; (+): Positive.

detection of MBL production in clinical isolates [28].

Step 1: Soak paper discs within a 0.1 M EDTA solution.

C for 18-24h.

**Figure 3.** Preparation of the imipenem-EDTA synergy test.

Step 2: Make a 0.5 McFarland bacterial suspension.

EDTA (ethylene-diamine-tetraacetic acid) is a polyamino carboxylic acid that binds metal ions like zinc and can inactivate the metallo-beta-lactamases. Therefore, it is used for the phenotypic

Step 3: Inoculate with a sterile cotton swab and place an imipenem and a ceftazidime disc at

Step 4: Place the EDTA discs at both sides in respect to the antibiotics as shown in Figure 3.

**3.2. Imipenem-EDTA synergy test**

*3.2.1. Procedure*

142 Trends in Infectious Diseases

the center of the plate.

Step 5: Incubate at 37o

Step 1: Make a 0.5 McFarland bacterial suspension.

Step 2: Inoculate with a sterile cotton swab and place two meropenem discs (Figure 5).

**Figure 4.** Interpretation of the imipenem-EDTA synergy test. (-): Negative; (+): Positive. Note that the upper left isolate is negative for MBL production but shows positive D-test between imipenem and ceftazidime indicating for the pres‐ ence of inducible AmpC beta-lactamases (The D-test is described in paragraph 3.6).

Step 3: Add 20 μl of phenylboronic acid 20 g/L on one of the two meropenem discs.

Step 4: Incubate at 37o C for 18-24h.

#### *3.3.2. Interpretation*

In case of KPC production, the phenylboronic acid that has been added to the second mero‐ penem disc will block the hydrolytic activity of the enzyme. As a consequence, the second disc will have a larger inhibition halo. The test is considered positive when the inhibition zone of the meropenem+phenylboronic acid is ≥ 5mm larger than the inhibition zone of meropenem alone (Figure 6).

Step 2: Inoculate with a sterile cotton swab and place a carbapenem disc at the center of the

Phenotypic and Molecular Methods for the Detection of Antibiotic Resistance Mechanisms in…

http://dx.doi.org/10.5772/57582

145

Step 3: Streak 3-5 colonies of the test isolate from the center to the periphery of the plate (Figure

The presence of a distorted inhibition zone due to growth of the indicator strain toward the carbapenem disc is interpreted as a positive result (Figure 8). This occurs due to carbapenemase production by the study isolate. Uncertain results need to be confirmed by other tests or

This test is a combination of the EDTA and the boronic acid test in a single plate and has been introduced in Greece after the emergence of Gram negative isolates co-producing KPC and MBL carbapenemases [33-37]. The advantage of the test is that it discriminates between carbapenem-susceptible, KPC-producing, MBL-producing and double-carbapenemase-

plate.

7).

Step 4: Incubate at 37o

**Figure 7.** Preparation of the Hodge test.

**3.5. Combination meropenem disc test**

*3.4.2. Interpretation*

molecular methods.

producing bacteria.

C for 18-24h.

**Figure 5.** Preparation of the boronic acid test.

**Figure 6.** Interpretation of the boronic acid test. (+): Positive; (-): Negative.

#### **3.4. Hodge test**

The Hodge test is used to reveal carbapenemase production [32]. This is achieved by inocu‐ lating the study isolate together with a carbapenem-susceptible indicator strain and evaluating the distortion of the indicator strain's inhibition zone because of carbapenemase production by the study isolate. Despite its usefulness, this test presents a disadvantage: it detects the presence of carbapenemases only, without being able to discriminate between different carbapenemase types (KPC or MBLs).

#### *3.4.1. Procedure*

Step 1: Make a 0.5 McFarland suspension of the indicator strain (for example *E. coli* ATCC 25922).

Step 2: Inoculate with a sterile cotton swab and place a carbapenem disc at the center of the plate.

Step 3: Streak 3-5 colonies of the test isolate from the center to the periphery of the plate (Figure 7).

Step 4: Incubate at 37o C for 18-24h.

**Figure 7.** Preparation of the Hodge test.

#### *3.4.2. Interpretation*

**Figure 6.** Interpretation of the boronic acid test. (+): Positive; (-): Negative.

The Hodge test is used to reveal carbapenemase production [32]. This is achieved by inocu‐ lating the study isolate together with a carbapenem-susceptible indicator strain and evaluating the distortion of the indicator strain's inhibition zone because of carbapenemase production by the study isolate. Despite its usefulness, this test presents a disadvantage: it detects the presence of carbapenemases only, without being able to discriminate between different

Step 1: Make a 0.5 McFarland suspension of the indicator strain (for example *E. coli* ATCC

**3.4. Hodge test**

*3.4.1. Procedure*

25922).

carbapenemase types (KPC or MBLs).

**Figure 5.** Preparation of the boronic acid test.

144 Trends in Infectious Diseases

The presence of a distorted inhibition zone due to growth of the indicator strain toward the carbapenem disc is interpreted as a positive result (Figure 8). This occurs due to carbapenemase production by the study isolate. Uncertain results need to be confirmed by other tests or molecular methods.

#### **3.5. Combination meropenem disc test**

This test is a combination of the EDTA and the boronic acid test in a single plate and has been introduced in Greece after the emergence of Gram negative isolates co-producing KPC and MBL carbapenemases [33-37]. The advantage of the test is that it discriminates between carbapenem-susceptible, KPC-producing, MBL-producing and double-carbapenemaseproducing bacteria.

will not present significant differences (≥5mm) from the meropenem disc alone. In case of KPC production, an increase of ≥5mm in the discs that are supplemented with boronic acid will be observed. MBL production will become evident by an increase of ≥5mm in the discs that are supplemented with EDTA. In case of a KPC+MBL-producer, the fourth disc will present the larger zone diameter of all. The EDTA-supplemented and boronic acid-supplemented discs may or may not have a ≥5mm larger zone diameter than that of the meropenem disc alone.

Phenotypic and Molecular Methods for the Detection of Antibiotic Resistance Mechanisms in…

http://dx.doi.org/10.5772/57582

147

Recently, a novel variation of this test has been proposed [38] for surveillance cultures from rectal swabs. The same principle is generally followed, except that each swab is initially suspended in 1 ml sterile saline by rotating and agitating it to release the microorganisms. Afterwards, the suspension is cultured onto McConkey agar using a different swab. This method allows the identification and differentiation of carbapenemase-producing *Enterobac‐*

**Figure 11.** Application of the combination meropenem disc test for the direct differentiation of carbapenemase-pro‐

**Figure 10.** Interpretation of the combination meropenem disc test.

*teriaceae* (Figure 11) directly at patient admission.

ducing *Enterobacteriaceae* in rectal swabs.

**Figure 8.** Interpretation of the Hodge test.

#### *3.5.1. Procedure*

Step 1: Make a 0.5 McFarland bacterial suspension.

Step 2: Inoculate with a sterile cotton swab and place four meropenem discs (Figure 9).

Step 3: Add 10 μl EDTA 0.1 M on the second disc, 20 μl of phenylboronic acid 20 g/L on the third disc and 20 μl of phenylboronic acid 20 g/L+10 μl EDTA 0.1 M on the fourth disc.

Step 4: Incubate at 37o C for 18-24h.

**Figure 9.** Preparation of the combination meropenem disc test.

#### *3.5.2. Interpretation*

The interpretation of the combination meropenem disc test is based on the comparison between the inhibition zones of the four meropenem discs as presented in Figure 10. If no carbapenemase is present, the zone diameters of the discs where inhibitors have been added will not present significant differences (≥5mm) from the meropenem disc alone. In case of KPC production, an increase of ≥5mm in the discs that are supplemented with boronic acid will be observed. MBL production will become evident by an increase of ≥5mm in the discs that are supplemented with EDTA. In case of a KPC+MBL-producer, the fourth disc will present the larger zone diameter of all. The EDTA-supplemented and boronic acid-supplemented discs may or may not have a ≥5mm larger zone diameter than that of the meropenem disc alone.

**Figure 10.** Interpretation of the combination meropenem disc test.

*3.5.1. Procedure*

146 Trends in Infectious Diseases

**Figure 8.** Interpretation of the Hodge test.

Step 4: Incubate at 37o

*3.5.2. Interpretation*

Step 1: Make a 0.5 McFarland bacterial suspension.

C for 18-24h.

**Figure 9.** Preparation of the combination meropenem disc test.

Step 2: Inoculate with a sterile cotton swab and place four meropenem discs (Figure 9).

Step 3: Add 10 μl EDTA 0.1 M on the second disc, 20 μl of phenylboronic acid 20 g/L on the third disc and 20 μl of phenylboronic acid 20 g/L+10 μl EDTA 0.1 M on the fourth disc.

The interpretation of the combination meropenem disc test is based on the comparison between the inhibition zones of the four meropenem discs as presented in Figure 10. If no carbapenemase is present, the zone diameters of the discs where inhibitors have been added Recently, a novel variation of this test has been proposed [38] for surveillance cultures from rectal swabs. The same principle is generally followed, except that each swab is initially suspended in 1 ml sterile saline by rotating and agitating it to release the microorganisms. Afterwards, the suspension is cultured onto McConkey agar using a different swab. This method allows the identification and differentiation of carbapenemase-producing *Enterobac‐ teriaceae* (Figure 11) directly at patient admission.

**Figure 11.** Application of the combination meropenem disc test for the direct differentiation of carbapenemase-pro‐ ducing *Enterobacteriaceae* in rectal swabs.

## **3.6. D-test**

The D-test is used for the detection of inducible AmpC beta-lactamases [39]. An antibiotic is used as an inducer for AmpC production (imipenem or cefoxitin) whereas others are used as substrates (ceftazidime, cefotaxime, piperacillin/tazobactam).

**3.7. CCCP test**

isolate [40].

**Figure 13.** Interpretation of the D-test.

*3.7.1. Procedure*

shown in Figure 15.

Step 5: Incubate at 37o

**Figure 14.** Incorporation of the D-test in a common antibiogram.

Step 2: Make a 0.5 McFarland bacterial suspension.

Step 1: Prepare agar plates containing CCCP at a concentration of 12.5 μM.

Step 4: Place a meropenem disc on both plates for each inoculation.

C for 18-24h.

Step 3: Inoculate with a sterile cotton swab on a CCCP-supplemented plate and in parallel on a CCCP-free plate. For economy reasons, two isolates may be inoculated on the same plate as

CCCP (Carbonyl cyanide m-chlorophenyl hydrazone) is an efflux pump inhibitor that can be added in Mueller-Hinton agar during its preparation. The test is used to detect efflux pump overexpression that contributes to or determines carbapenem resistance in the study

Phenotypic and Molecular Methods for the Detection of Antibiotic Resistance Mechanisms in…

http://dx.doi.org/10.5772/57582

149

#### *3.6.1. Procedure*

Step 1: Make a 0.5 McFarland bacterial suspension.

Step 2: Inoculate with a sterile cotton swab and place an imipenem disc.

Step 3: Place substrate discs (for example ceftazidime and piperacillin/tazobactam) near the imipenem disc as shown in Figure 12.

Step 4: Incubate at 37o C for 18-24h.

#### *3.6.2. Interpretation*

The test is positive when a D-shaped inhibition zone is observed for one of the substrate discs (Figure 13) because of the imipenem-mediated induction of the AmpC production and the subsequent inactivation of the substrate antibiotic by the beta-lactamase. An important advantage of the test is that it can be easily incorporated within any routine antibiogram as shown in Figure 14.
